Combination of antibody-drug conjugate and immune checkpoint inhibitor

ABSTRACT

A pharmaceutical composition and a therapeutic method wherein an antibody-drug conjugate and an immune checkpoint inhibitor are administered in combination, and the antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker represented by the following formula (wherein A represents the connecting position to an antibody) is conjugated to the antibody via a thioether bond; and a pharmaceutical composition and a therapeutic method for use in treatment of a disease that can be ameliorated through an antitumor immunity-activating effect wherein the antibody-drug conjugate is included.

RELATED APPLICATIONS

The present application claims priority under 37 U.S.C. § 371 toInternational Patent Application No. PCT/JP2017/044426, filed Dec. 11,2017, which claims priority to and the benefit of Japanese PatentApplication Nos. 2016-240442, filed on Dec. 12, 2016, 2017-097067, filedon May 16, 2017, and 2017-183149, filed on Sep. 25, 2017. The contentsof these applications are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, is named 111119-0124 SL.txtand is 7 kb in size.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition and atherapeutic method wherein a specific antibody-drug conjugate and animmune checkpoint inhibitor are administered in combination, and apharmaceutical composition and a therapeutic method for use in treatmentof a disease that can be ameliorated through an antitumorimmunity-activating effect wherein a specific antibody-drug conjugate isincluded.

BACKGROUND ART

An antibody-drug conjugate (ADC) having a drug with cytotoxicityconjugated to an antibody, whose antigen is expressed on the surface ofcancer cells and which also binds to an antigen capable of cellularinternalization, and therefore can deliver the drug selectively tocancer cells, is thus expected to cause accumulation of the drug withincancer cells and to kill the cancer cells (Non-Patent Literatures 1 to5).

As one of such antibody-drug conjugates, an antibody-drug conjugateincluding an antibody and exatecan, which is a topoisomerase Iinhibitor, as components is known (Patent Literatures 1 to 7). Amongthese, anti-HER2 antibody-drug conjugates (Non-Patent Literatures 6, 7),which exert a particularly superior antitumor effect and safety, arecurrently under clinical studies.

Immune checkpoint inhibitors are agents that inhibit the immunesuppression system and activate antitumor immunity (Non-PatentLiteratures 8 to 10). Known examples of immune checkpoint inhibitorsinclude nivolumab (Patent Literature 8) and pembrolizumab (PatentLiterature 9) each of which is an anti-PD-1 antibody; atezolizumab(Patent Literature 10), durvalumab (Patent Literature 11), and avelumab(Patent Literature 12), each of which is an anti-PD-L1 antibody; andipilimumab (Patent Literature 13) and tremelimumab (Patent Literature14), each of which is an anti-CTLA-4 antibody.

As a case in which an antibody-drug conjugate and an immune checkpointinhibitor are administered in combination, a study on the use oftrastuzumab emtansine (T-DM1) and an anti-CTLA-4/PD-1 antibody incombination is known (Non-Patent Literature 11).

CITATION LIST Patent Literatures

-   Patent Literature 1: International Publication No. WO 2014/057687-   Patent Literature 2: International Publication No. WO 2014/061277-   Patent Literature 3: International Publication No. WO 2015/098099-   Patent Literature 4: International Publication No. WO 2015/115091-   Patent Literature 5: International Publication No. WO 2015/146132-   Patent Literature 6: International Publication No. WO 2015/155976-   Patent Literature 7: International Publication No. WO 2015/155998-   Patent Literature 8: International Publication No. WO 2006/121168-   Patent Literature 9: International Publication No. WO 2008/156712-   Patent Literature 10: International Publication No. WO 2010/077634-   Patent Literature 11: International Publication No. WO 2011/066389-   Patent Literature 12: International Publication No. WO 2013/079174-   Patent Literature 13: International Publication No. WO 2001/014424-   Patent Literature 14: International Publication No. WO 2000/037504

Non-Patent Literatures

-   Non-Patent Literature 1: Ducry, L., et al., Bioconjugate    Chem. (2010) 21, 5-13.-   Non-Patent Literature 2: Alley, S. C., et al., Current Opinion in    Chemical Biology (2010) 14, 529-537.-   Non-Patent Literature 3: Damle N. K. Expert Opin. Biol. Ther. (2004)    4, 1445-1452.-   Non-Patent Literature 4: Senter P. D., et al., Nature    Biotechnology (2012) 30, 631-637.-   Non-Patent Literature 5: Burris, III et al., J Clin Oncol 29:    398-405.-   Non-Patent Literature 6: Ogitani Y. et al., Clinical Cancer    Research (2016) 22(20), 5097-5108.-   Non-Patent Literature 7: Ogitani Y. et al., Cancer Science (2016)    107, 1039-1046.-   Non-Patent Literature 8: Menon S. et al., Cancers (2016) 8, 106.-   Non-Patent Literature 9: Pardoll D M., Nat Rev Cancer (2012) 12,    252-264.-   Non-Patent Literature 10: Wolchok J D., Cell (2015) 162, 937.-   Non-Patent Literature 11: Muller P. et al., Science Translational    Medicine (2015) 7(315), 315ra188.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a pharmaceuticalcomposition and a therapeutic method which exert a particularly superiorantitumor effect and safety through administering an antibody-drugconjugate and an immune checkpoint inhibitor in combination. Anotherobject of the present invention is to provide a pharmaceuticalcomposition and a therapeutic method for use in treatment of a diseasethat can be ameliorated through an antitumor immunity-activating effectwherein a specific antibody-drug conjugate is included.

Solution to Problem

The present inventors found that an excellent antitumor effect isexerted through administering a specific antibody-drug conjugate and animmune checkpoint inhibitor in combination; and further found that theantibody-drug conjugate has an antitumor immunity-activating effect.

Specifically, the present invention relates to the following.

[1] A pharmaceutical composition wherein an antibody-drug conjugate andan immune checkpoint inhibitor are administered in combination, and theantibody-drug conjugate is an antibody-drug conjugate in which adrug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond.[2] The pharmaceutical composition according to [1], wherein theantibody in the antibody-drug conjugate is an anti-HER2 antibody, ananti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.[3] The pharmaceutical composition according to [2], wherein theantibody in the antibody-drug conjugate is an anti-HER2 antibody.[4] The pharmaceutical composition according to [2] or [3], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofan amino acid sequence consisting of amino acid residues 1 to 449 of SEQID NO: 1 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[5] The pharmaceutical composition according to [2] or [3], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofthe amino acid sequence represented by SEQ ID NO: 1 and a light chainconsisting of the amino acid sequence represented by SEQ ID NO: 2.[6] The pharmaceutical composition according to any one of [1] to [5],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from2 to 8.[7] The pharmaceutical composition according to any one of [1] to [5],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7 to 8.[8] The pharmaceutical composition according to any one of [1] to [5],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7.5 to 8.[9] The pharmaceutical composition according to any one of [1] to [8],wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, ananti-PD-L1 antibody, or an anti-CTLA-4 antibody.[10] The pharmaceutical composition according to [9], wherein the immunecheckpoint inhibitor is an anti-PD-1 antibody.[11] The pharmaceutical composition according to [9], wherein the immunecheckpoint inhibitor is an anti-PD-L1 antibody.[12] The pharmaceutical composition according to [9], wherein the immunecheckpoint inhibitor is an anti-CTLA-4 antibody.[13] The pharmaceutical composition according to any one of [1] to [12],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare separately contained as active components in different formulations,and are administered simultaneously or at different times.[14] The pharmaceutical composition according to any one of [1] to [12],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare contained as active components in a single formulation andadministered.[15] The pharmaceutical composition according to any one of [1] to [14],wherein the composition is for treating cancer.[16] The pharmaceutical composition according to [15], wherein thecancer is at least one selected from the group consisting of lungcancer, urothelial cancer, colorectal cancer, prostate cancer, ovariancancer, pancreatic cancer, breast cancer, bladder cancer, gastriccancer, esophagogastric junction adenocarcinoma, gastrointestinalstromal tumor, uterine cervix cancer, esophageal cancer, squamous cellcarcinoma, peritoneal cancer, liver cancer, hepatocellular cancer,endometrial cancer, uterine cancer, salivary gland cancer, kidneycancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignantlymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nervesheath tumor, head-and-neck cancer, skin cancer, pharyngeal cancer,gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, andsarcoma.[17] The pharmaceutical composition according to [16], wherein thecancer is colorectal cancer.[18] The pharmaceutical composition according to [16], wherein thecancer is breast cancer.[19] The pharmaceutical composition according to any one of [1] to [18],wherein the antibody-drug conjugate has an antitumor immunity-activatingeffect.[20] The pharmaceutical composition according to any one of [1] to [19],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[21] The pharmaceutical composition according to any one of [1] to [20],wherein the antibody-drug conjugate has a promoting effect on theformation of immune memory against tumor.

[22] The pharmaceutical composition according to [21], wherein the tumoris expressing an antigen for the antibody in the antibody-drugconjugate.

[23] The pharmaceutical composition according to [21], wherein a part ofthe cells of the tumor are not expressing an antigen for the antibody inthe antibody-drug conjugate.

[24] The pharmaceutical composition according to any one of [1] to [23],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[25] The pharmaceutical composition according to any one of [1] to [24],wherein the immune checkpoint inhibitor deactivates an immunosuppressionsignal generated through elevation of the expression level of PD-L1 oncancer cells promoted by the antibody-drug conjugate, and thereby theantibody-drug conjugate exhibits a higher antitumor effect.[26] A pharmaceutical composition for use in treatment of a disease thatcan be ameliorated through an antitumor immunity-activating effect,wherein the pharmaceutical composition contains an antibody-drugconjugate in which a drug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond.[27] The pharmaceutical composition according to [26], wherein theantibody-drug conjugate has at least one effect selected from the groupconsisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[28] The pharmaceutical composition according to [26] or [27], whereinthe antibody-drug conjugate has a promoting effect on the formation ofimmune memory against tumor.

[29] The pharmaceutical composition according to [28], wherein the tumoris expressing an antigen for the antibody in the antibody-drugconjugate.

[30] The pharmaceutical composition according to [28], wherein a part ofthe cells of the tumor are not expressing an antigen for the antibody inthe antibody-drug conjugate.

[31] The pharmaceutical composition according to any one of [26] to[30], wherein the antibody-drug conjugate has at least one effectselected from the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[32] The pharmaceutical composition according to any one of [26] to[31], wherein the antibody in the antibody-drug conjugate is ananti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or ananti-B7-H3 antibody.

[33] The pharmaceutical composition according to [32], wherein theantibody in the antibody-drug conjugate is an anti-HER2 antibody.

[34] The pharmaceutical composition according to [32] or [33], whereinthe anti-HER2 antibody is an antibody comprising a heavy chainconsisting of an amino acid sequence consisting of amino acid residues 1to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acidsequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[35] The pharmaceutical composition according to [32] or [33], whereinthe anti-HER2 antibody is an antibody comprising a heavy chainconsisting of the amino acid sequence represented by SEQ ID NO: 1 and alight chain consisting of the amino acid sequence represented by SEQ IDNO: 2.[36] The pharmaceutical composition according to any one of [26] to[35], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 2 to 8.[37] The pharmaceutical composition according to any one of [26] to[35], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 7 to 8.[38] The pharmaceutical composition according to any one of [26] to[35], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 7.5 to 8.[39] The pharmaceutical composition according to any one of [26] to[38], wherein the disease is at least one selected from the groupconsisting of lung cancer, urothelial cancer, colorectal cancer,prostate cancer, ovarian cancer, pancreatic cancer, breast cancer,bladder cancer, gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[40] The pharmaceutical composition according to [39], wherein thedisease is colorectal cancer.[41] The pharmaceutical composition according to [39], wherein thedisease is breast cancer.[42] A pharmaceutical composition for use in treatment of a disease thatcan be ameliorated through an antitumor immunity-activating effect,wherein the pharmaceutical composition releases the compound representedby the following formula:

in a tumor.[43] The pharmaceutical composition according to [42], wherein thecompound has at least one effect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[44] The pharmaceutical composition according to [42] or [43], whereinthe compound has a promoting effect on the formation of immune memoryagainst tumor.

[45] The pharmaceutical composition according to any one of [42] to[44], wherein the compound has at least one effect selected from thegroup consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[46] The pharmaceutical composition according to any one of [42] to[45], wherein the disease is at least one selected from the groupconsisting of lung cancer, urothelial cancer, colorectal cancer,prostate cancer, ovarian cancer, pancreatic cancer, breast cancer,bladder cancer, gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[47] A therapeutic method wherein an antibody-drug conjugate and animmune checkpoint inhibitor are administered in combination, and theantibody-drug conjugate is an antibody-drug conjugate in which adrug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond.[48] The therapeutic method according to [47], wherein the antibody inthe antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.[49] The therapeutic method according to [48], wherein the antibody inthe antibody-drug conjugate is an anti-HER2 antibody.[50] The therapeutic method according to [48] or [49], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofan amino acid sequence consisting of amino acid residues 1 to 449 of SEQID NO: 1 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[51] The therapeutic method according to [48] or [49], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofthe amino acid sequence represented by SEQ ID NO: 1 and a light chainconsisting of the amino acid sequence represented by SEQ ID NO: 2.[52] The therapeutic method according to any one of [47] to [51],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from2 to 8.[53] The therapeutic method according to any one of [47] to [51],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7 to 8.[54] The therapeutic method according to any one of [47] to [51],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7.5 to 8.[55] The therapeutic method according to any one of [47] to [54],wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, ananti-PD-L1 antibody, or an anti-CTLA-4 antibody.[56] The therapeutic method according to [55], wherein the immunecheckpoint inhibitor is an anti-PD-1 antibody.[57] The therapeutic method according to [55], wherein the immunecheckpoint inhibitor is an anti-PD-L1 antibody.[58] The therapeutic method according to [55], wherein the immunecheckpoint inhibitor is an anti-CTLA-4 antibody.[59] The therapeutic method according to any one of [47] to [58],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare separately contained as active components in different formulations,and are administered simultaneously or at different times.[60] The therapeutic method according to any one of [47] to [58],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare contained as active components in a single formulation andadministered.[61] The therapeutic method according to any one of [47] to [60],wherein the therapeutic method is for treating cancer.[62] The therapeutic method according to [61], wherein the cancer is atleast one selected from the group consisting of lung cancer, urothelialcancer, colorectal cancer, prostate cancer, ovarian cancer, pancreaticcancer, breast cancer, bladder cancer, gastric cancer, esophagogastricjunction adenocarcinoma, gastrointestinal stromal tumor, uterine cervixcancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer,liver cancer, hepatocellular cancer, endometrial cancer, uterine cancer,salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer,penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[63] The therapeutic method according to [62], wherein the cancer iscolorectal cancer.[64] The therapeutic method according to [62], wherein the cancer isbreast cancer.[65] The therapeutic method according to any one of [47] to [64],wherein the antibody-drug conjugate has an antitumor immunity-activatingeffect.[66] The pharmaceutical composition according to any one of [47] to[65], wherein the antibody-drug conjugate has at least one effectselected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[67] The therapeutic method according to any one of [47] to [66],wherein the antibody-drug conjugate has a promoting effect on theformation of immune memory against tumor.

[68] The therapeutic method according to [67], wherein the tumor isexpressing an antigen for the antibody in the antibody-drug conjugate.

[69] The therapeutic method according to [67], wherein a part of thecells of the tumor are not expressing an antigen for the antibody in theantibody-drug conjugate.

[70] The therapeutic method according to any one of [47] to [69],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[71] The therapeutic method according to any one of [47] to [70],wherein the immune checkpoint inhibitor deactivates an immunosuppressionsignal generated through elevation of the expression level of PD-L1 oncancer cells promoted by the antibody-drug conjugate, and thereby theantibody-drug conjugate exhibits a higher antitumor effect.[72] A therapeutic method for use in treatment of a disease that can beameliorated through an antitumor immunity-activating effect wherein anantibody-drug conjugate in which a drug-linker represented by thefollowing formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond, is administered.[73] The therapeutic method according to [72], wherein the antibody-drugconjugate has at least one effect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[74] The therapeutic method according to [72] or [73], wherein theantibody-drug conjugate has a promoting effect on the formation ofimmune memory against tumor.

[75] The therapeutic method according to [74], wherein the tumor isexpressing an antigen for the antibody in the antibody-drug conjugate.

[76] The therapeutic method according to [74], wherein a part of thecells of the tumor are not expressing an antigen for the antibody in theantibody-drug conjugate.

[77] The therapeutic method according to any one of [72] to [76],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[78] The therapeutic method according to any one of [72] to [77],wherein the antibody in the antibody-drug conjugate is an anti-HER2antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or ananti-B7-H3 antibody.

[79] The therapeutic method according to [78], wherein the antibody inthe antibody-drug conjugate is an anti-HER2 antibody.

[80] The therapeutic method according to [78] or [79], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofan amino acid sequence consisting of amino acid residues 1 to 449 of SEQID NO: 1 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[81] The therapeutic method according to [78] or [79], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofthe amino acid sequence represented by SEQ ID NO: 1 and a light chainconsisting of the amino acid sequence represented by SEQ ID NO: 2.[82] The therapeutic method according to any one of [72] to [81],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from2 to 8.[83] The therapeutic method according to any one of [72] to [81],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7 to 8.[84] The therapeutic method according to any one of [72] to [81],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7.5 to 8.[85] The therapeutic method according to any one of [72] to [84],wherein the disease is at least one selected from the group consistingof lung cancer, urothelial cancer, colorectal cancer, prostate cancer,ovarian cancer, pancreatic cancer, breast cancer, bladder cancer,gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[86] The therapeutic method according to [85], wherein the disease iscolorectal cancer.[87] The therapeutic method according to [85], wherein the disease isbreast cancer.[88] A therapeutic method for use in treatment of a disease that can beameliorated through an antitumor immunity-activating effect, wherein thetherapeutic method releases the compound represented by the followingformula:

in a tumor.[89] The therapeutic method according to [88], wherein the compound hasat least one effect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[90] The therapeutic method according to [88] or [89], wherein thecompound has a promoting effect on the formation of immune memoryagainst tumor.

[91] The therapeutic method according to any one of [88] to [90],wherein the compound has at least one effect selected from the groupconsisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[92] The therapeutic method according to any one of [88] to [91],wherein the disease is at least one selected from the group consistingof lung cancer, urothelial cancer, colorectal cancer, prostate cancer,ovarian cancer, pancreatic cancer, breast cancer, bladder cancer,gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[93] An antibody-drug conjugate for treating a disease through beingadministered in combination with an immune checkpoint inhibitor, whereina drug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond in the antibody-drugconjugate.[94] The antibody-drug conjugate according to [93], wherein the antibodyin the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.[95] The antibody-drug conjugate according to [94], wherein the antibodyin the antibody-drug conjugate is an anti-HER2 antibody.[96] The antibody-drug conjugate according to [94] or [95], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofan amino acid sequence consisting of amino acid residues 1 to 449 of SEQID NO: 1 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[97] The antibody-drug conjugate according to [94] or [95], wherein theanti-HER2 antibody is an antibody comprising a heavy chain consisting ofthe amino acid sequence represented by SEQ ID NO: 1 and a light chainconsisting of the amino acid sequence represented by SEQ ID NO: 2.[98] The antibody-drug conjugate according to any one of [93] to [97],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from2 to 8.[99] The antibody-drug conjugate according to any one of [93] to [97],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7 to 8.[100] The antibody-drug conjugate according to any one of [93] to [97],wherein the average number of units of the drug-linker conjugated perantibody molecule in the antibody-drug conjugate is in the range of from7.5 to 8.[101] The antibody-drug conjugate according to any one of [93] to [100],wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, ananti-PD-L1 antibody, or an anti-CTLA-4 antibody.[102] The antibody-drug conjugate according to [101], wherein the immunecheckpoint inhibitor is an anti-PD-1 antibody.[103] The antibody-drug conjugate according to [101], wherein the immunecheckpoint inhibitor is an anti-PD-L1 antibody.[104] The antibody-drug conjugate according to [101], wherein the immunecheckpoint inhibitor is an anti-CTLA-4 antibody.[105] The antibody-drug conjugate according to any one of [93] to [104],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare separately contained as active components in different formulations,and are administered simultaneously or at different times.[106] The antibody-drug conjugate according to any one of [93] to [104],wherein the antibody-drug conjugate and the immune checkpoint inhibitorare contained as active components in a single formulation andadministered.[107] The antibody-drug conjugate according to any one of [93] to [106],wherein the antibody-drug conjugate is for treating cancer.[108] The antibody-drug conjugate according to [107], wherein the canceris at least one selected from the group consisting of lung cancer,urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer,pancreatic cancer, breast cancer, bladder cancer, gastric cancer,esophagogastric junction adenocarcinoma, gastrointestinal stromal tumor,uterine cervix cancer, esophageal cancer, squamous cell carcinoma,peritoneal cancer, liver cancer, hepatocellular cancer, endometrialcancer, uterine cancer, salivary gland cancer, kidney cancer, vulvalcancer, thyroid cancer, penis cancer, leukemia, malignant lymphoma,plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor,head-and-neck cancer, skin cancer, pharyngeal cancer, gallbladdercancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.[109] The antibody-drug conjugate according to [108], wherein the canceris colorectal cancer.[110] The antibody-drug conjugate according to [108], wherein the canceris breast cancer.[111] The antibody-drug conjugate according to any one of [93] to [110],wherein the antibody-drug conjugate has an antitumor immunity-activatingeffect.[112] The antibody-drug conjugate according to any one of [93] to [111],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[113] The antibody-drug conjugate according to any one of [93] to [112],wherein the antibody-drug conjugate has a promoting effect on theformation of immune memory against tumor.

[114] The antibody-drug conjugate according to [113], wherein the tumoris expressing an antigen for the antibody in the antibody-drugconjugate.

[115] The antibody-drug conjugate according to [113], wherein a part ofthe cells of the tumor are not expressing an antigen for the antibody inthe antibody-drug conjugate.

[116] The antibody-drug conjugate according to any one of [93] to [115],wherein the antibody-drug conjugate has at least one effect selectedfrom the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[117] The antibody-drug conjugate according to any one of [93] to [116],wherein the immune checkpoint inhibitor deactivates an immunosuppressionsignal generated through elevation of the expression level of PD-L1 oncancer cells promoted by the antibody-drug conjugate, and thereby theantibody-drug conjugate exhibits a higher antitumor effect.[118] An antibody-drug conjugate for use in treatment of a disease thatcan be ameliorated through an antitumor immunity-activating effect,wherein a drug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,

is conjugated to the antibody via a thioether bond.

[119] The antibody-drug conjugate according to [118], wherein theantibody-drug conjugate has at least one effect selected from the groupconsisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[120] The antibody-drug conjugate according to [118] or [119], whereinthe antibody-drug conjugate has a promoting effect on the formation ofimmune memory against tumor.

[121] The antibody-drug conjugate according to [120], wherein the tumoris expressing an antigen for the antibody in the antibody-drugconjugate.

[122] The antibody-drug conjugate according to [120], wherein a part ofthe cells of the tumor are not expressing an antigen for the antibody inthe antibody-drug conjugate.

[123] The antibody-drug conjugate according to any one of [118] to[122], for use in treatment of a disease that can be ameliorated throughat least one effect selected from the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[124] The antibody-drug conjugate according to any one of [118] to[123], wherein the antibody in the antibody-drug conjugate is ananti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or ananti-B7-H3 antibody.

[125] The antibody-drug conjugate according to [124], wherein theantibody in the antibody-drug conjugate is an anti-HER2 antibody.

[126] The antibody-drug conjugate according to [124] or [125], whereinthe anti-HER2 antibody is an antibody comprising a heavy chainconsisting of an amino acid sequence consisting of amino acid residues 1to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acidsequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2.[127] The antibody-drug conjugate according to [124] or [125], whereinthe anti-HER2 antibody is an antibody comprising a heavy chainconsisting of the amino acid sequence represented by SEQ ID NO: 1 and alight chain consisting of the amino acid sequence represented by SEQ IDNO: 2.[128] The antibody-drug conjugate according to any one of [118] to[127], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 2 to 8.[129] The antibody-drug conjugate according to any one of [118] to[127], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 7 to 8.[130] The antibody-drug conjugate according to any one of [118] to[127], wherein the average number of units of the drug-linker conjugatedper antibody molecule in the antibody-drug conjugate is in the range offrom 7.5 to 8.[131] The antibody-drug conjugate according to any one of [118] to[130], wherein the disease is at least one selected from the groupconsisting of lung cancer, urothelial cancer, colorectal cancer,prostate cancer, ovarian cancer, pancreatic cancer, breast cancer,bladder cancer, gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[132] The antibody-drug conjugate according to [131], wherein thedisease is colorectal cancer.[133] The antibody-drug conjugate according to [131], wherein thedisease is breast cancer.[134] A compound for use in treatment of a disease that can beameliorated through an antitumor immunity-activating effect, wherein thecompound is represented by the following formula:

and released in a tumor.

The compound according to [134], wherein the compound has at least oneeffect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[136] The compound according to [134] or [135], wherein the compound hasa promoting effect on the formation of immune memory against tumor.

[137] The compound according to any one of [134] to [136], wherein thecompound has at least one effect selected from the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[138] The compound according to any one of [134] to [137], wherein thedisease is at least one selected from the group consisting of lungcancer, urothelial cancer, colorectal cancer, prostate cancer, ovariancancer, pancreatic cancer, breast cancer, bladder cancer, gastriccancer, esophagogastric junction adenocarcinoma, gastrointestinalstromal tumor, uterine cervix cancer, esophageal cancer, squamous cellcarcinoma, peritoneal cancer, liver cancer, hepatocellular cancer,endometrial cancer, uterine cancer, salivary gland cancer, kidneycancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignantlymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nervesheath tumor, head-and-neck cancer, skin cancer, pharyngeal cancer,gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, andsarcoma.[139] Use of an antibody-drug conjugate for production of a medicine fortreating a disease through being administered in combination with animmune checkpoint inhibitor, wherein a drug-linker represented by thefollowing formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond in the antibody-drugconjugate.[140] The use according to [139], wherein the antibody in theantibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody,an anti-TROP2 antibody, or an anti-B7-H3 antibody.[141] The use according to [140], wherein the antibody in theantibody-drug conjugate is an anti-HER2 antibody.[142] The use according to [140] or [141], wherein the anti-HER2antibody is an antibody comprising a heavy chain consisting of an aminoacid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1and a light chain consisting of an amino acid sequence consisting ofamino acid residues 1 to 214 of SEQ ID NO: 2.[143] The use according to [140] or [141], wherein the anti-HER2antibody is an antibody comprising a heavy chain consisting of the aminoacid sequence represented by SEQ ID NO: 1 and a light chain consistingof the amino acid sequence represented by SEQ ID NO: 2.[144] The use according to any one of [139] to [143], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 2 to 8.[145] The use according to any one of [139] to [143], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 7 to 8.[146] The use according to any one of [139] to [143], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 7.5 to8.[147] The use according to any one of [139] to [146], wherein the immunecheckpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody,or an anti-CTLA-4 antibody.[148] The use according to [147], wherein the immune checkpointinhibitor is an anti-PD-1 antibody.[149] The use according to [147], wherein the immune checkpointinhibitor is an anti-PD-L1 antibody.[150] The use according to [147], wherein the immune checkpointinhibitor is an anti-CTLA-4 antibody.[151] The use according to any one of [139] to [150], wherein theantibody-drug conjugate and the immune checkpoint inhibitor areseparately contained as active components in different formulations, andare administered simultaneously or at different times.[152] The use according to any one of [139] to [150], wherein theantibody-drug conjugate and the immune checkpoint inhibitor arecontained as active components in a single formulation and administered.[153] The use according to any one of [139] to [152], wherein the use isfor treating cancer.[154] The use according to [153], wherein the cancer is at least oneselected from the group consisting of lung cancer, urothelial cancer,colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer,breast cancer, bladder cancer, gastric cancer, esophagogastric junctionadenocarcinoma, gastrointestinal stromal tumor, uterine cervix cancer,esophageal cancer, squamous cell carcinoma, peritoneal cancer, livercancer, hepatocellular cancer, endometrial cancer, uterine cancer,salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer,penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.[155] The use according to [154], wherein the cancer is colorectalcancer.[156] The use according to [154], wherein the cancer is breast cancer.[157] The use according to any one of [139] to [156], wherein theantibody-drug conjugate has an antitumor immunity-activating effect.[158] The use according to any one of [139] to [157], wherein theantibody-drug conjugate has at least one effect selected from the groupconsisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[159] The use according to any one of [139] to [158], wherein theantibody-drug conjugate has a promoting effect on the formation ofimmune memory against tumor.

[160] The use according to [159], wherein the tumor is expressing anantigen for the antibody in the antibody-drug conjugate.

[161] The use according to [159], wherein a part of the cells of thetumor are not expressing an antigen for the antibody in theantibody-drug conjugate.

[162] The use according to any one of [139] to [161], wherein theantibody-drug conjugate has at least one effect selected from the groupconsisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[163] The use according to any one of [139] to [162], wherein the immunecheckpoint inhibitor deactivates an immunosuppression signal generatedthrough elevation of the expression level of PD-L1 on cancer cellspromoted by the antibody-drug conjugate, and thereby the antibody-drugconjugate exhibits a higher antitumor effect.[164] Use of an antibody-drug conjugate for production of a medicine foruse in treatment of a disease that can be ameliorated through anantitumor immunity-activating effect, wherein a drug-linker representedby the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond in the antibody-drugconjugate.[165] The use according to [164], wherein the antibody-drug conjugatehas at least one effect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[166] The use according to [164] or [165], wherein the antibody-drugconjugate has a promoting effect on the formation of immune memoryagainst tumor.

[167] The use according to [166], wherein the tumor is expressing anantigen for the antibody in the antibody-drug conjugate.

[168] The use according to [166], wherein a part of the cells of thetumor are not expressing an antigen for the antibody in theantibody-drug conjugate.

[169] The use according to any one of [164] to [168], wherein the use isfor production of a medicine for use in treatment of a disease that canbe ameliorated through at least one effect selected from the groupconsisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[170] The use according to any one of [164] to [169], wherein theantibody in the antibody-drug conjugate is an anti-HER2 antibody, ananti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[171] The use according to [170], wherein the antibody in theantibody-drug conjugate is an anti-HER2 antibody.

[172] The use according to [170] or [171], wherein the anti-HER2antibody is an antibody comprising a heavy chain consisting of an aminoacid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1and a light chain consisting of an amino acid sequence consisting ofamino acid residues 1 to 214 of SEQ ID NO: 2.[173] The use according to [170] or [171], wherein the anti-HER2antibody is an antibody comprising a heavy chain consisting of the aminoacid sequence represented by SEQ ID NO: 1 and a light chain consistingof the amino acid sequence represented by SEQ ID NO: 2.[174] The use according to any one of [164] to [173], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 2 to 8.[175] The use according to any one of [164] to [173], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 7 to 8.[176] The use according to any one of [164] to [173], wherein theaverage number of units of the drug-linker conjugated per antibodymolecule in the antibody-drug conjugate is in the range of from 7.5 to8.[177] The use according to any one of [164] to [176], wherein thedisease is at least one selected from the group consisting of lungcancer, urothelial cancer, colorectal cancer, prostate cancer, ovariancancer, pancreatic cancer, breast cancer, bladder cancer, gastriccancer, esophagogastric junction adenocarcinoma, gastrointestinalstromal tumor, uterine cervix cancer, esophageal cancer, squamous cellcarcinoma, peritoneal cancer, liver cancer, hepatocellular cancer,endometrial cancer, uterine cancer, salivary gland cancer, kidneycancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignantlymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nervesheath tumor, head-and-neck cancer, skin cancer, pharyngeal cancer,gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, andsarcoma.[178] The use according to [177], wherein the disease is colorectalcancer.[179] The use according to [177], wherein the disease is breast cancer.[180] Use of a compound for production of a medicine for use intreatment of a disease that can be ameliorated through an antitumorimmunity-activating effect, wherein the compound is represented by thefollowing formula:

and released in a tumor.[181] The use according to [180], wherein the compound has at least oneeffect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

[182] The use according to [180] or [181], wherein the compound has apromoting effect on the formation of immune memory against tumor.

[183] The use according to any one of [180] to [182], wherein thecompound has at least one effect selected from the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

[184] The use according to any one of [180] to [183], wherein thedisease is at least one selected from the group consisting of lungcancer, urothelial cancer, colorectal cancer, prostate cancer, ovariancancer, pancreatic cancer, breast cancer, bladder cancer, gastriccancer, esophagogastric junction adenocarcinoma, gastrointestinalstromal tumor, uterine cervix cancer, esophageal cancer, squamous cellcarcinoma, peritoneal cancer, liver cancer, hepatocellular cancer,endometrial cancer, uterine cancer, salivary gland cancer, kidneycancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignantlymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nervesheath tumor, head-and-neck cancer, skin cancer, pharyngeal cancer,gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, andsarcoma.

Advantageous Effects of Invention

The present invention can provide a pharmaceutical composition and atherapeutic method which exert a particularly superior antitumor effectand safety through administering a specific antibody-drug conjugate andan immune checkpoint inhibitor in combination. In addition, the presentinvention can provide a pharmaceutical composition and a therapeuticmethod for treating a disease that can be ameliorated through apromoting effect on the formation of immune memory against tumor whereina specific antibody-drug conjugate is included.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of a heavy chain of a humanizedanti-HER2 antibody (SEQ ID NO: 1).

FIG. 2 shows an amino acid sequence of a light chain of a humanizedanti-HER2 antibody (SEQ ID NO: 2).

FIG. 3 is a diagram showing life-prolonging effects of different agentson mice with subcutaneously transplanted CT26.WT-hHER2 cells. Comparisonwas made on life-prolonging effect between a single administration groupwith each of an antibody-drug conjugate (1) and an anti-PD-1 antibody(clone RMP1-14) and a combined administration group.

FIG. 4 is a diagram showing life-prolonging effects of different agentson mice with subcutaneously transplanted CT26.WT-hHER2 cells. Comparisonwas made on life-prolonging effect between a single administration groupwith each of an antibody-drug conjugate (1) and an anti-PD-1 antibody(clone RMP1-14) and a combined administration group.

FIG. 5 is a diagram showing transition of tumor volume in antibody-drugconjugate (1)-treated cured mice and control mice with subcutaneouslytransplanted (retransplanted) CT26.WT-hHER2 cells or CT26.WT-mock cells.

FIG. 6 is a diagram showing immune response to an antigen derived fromCT26.WT-hHER2 cells (the number of IFNγ-producing splenocytes) inantibody-drug conjugate (1)-treated cured mice and control mice withsubcutaneously transplanted (retransplanted) CT26.WT-hHER2 cells.

FIG. 7 is a diagram showing immune response to an antigen derived fromCT26.WT-mock cells (the number of IFNγ-producing splenocytes) inantibody-drug conjugate (1)-treated cured mice and control mice withsubcutaneously transplanted (retransplanted) CT26.WT-hHER2 cells.

FIG. 8 is a diagram showing immune response to an antigen derived fromCT26.WT-hHER2 cells (the number of IFNγ-producing splenocytes) inantibody-drug conjugate (1)-treated cured mice and control mice withsubcutaneously transplanted (retransplanted) CT26.WT-mock cells.

FIG. 9 is a diagram showing immune response to an antigen derived fromCT26.WT-mock cells (the number of IFNγ-producing splenocytes) inantibody-drug conjugate (1)-treated cured mice and control mice withsubcutaneously transplanted (retransplanted) CT26.WT-mock cells.

FIG. 10 is a series of diagrams showing expression levels of CD86 inbone marrow-derived dendritic cells treated with a compound (A) andthose treated with DMSO as determined with flow cytometry.

FIG. 11 is a series of diagrams showing expression levels of MHC classII in bone marrow-derived dendritic cells treated with a compound (A)and those treated with DMSO as determined with flow cytometry.

FIG. 12 is a diagram showing the number of dendritic cells amongintratumor lymphocytes as determined with flow cytometry for anantibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 13 is a diagram showing the number of CD86-positive cells amongintratumor dendritic cells as determined with flow cytometry for anantibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 14 is a diagram showing expression levels of CD86 on intratumordendritic cells as determined with flow cytometry and expressed as MFIfor an antibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 15 is a diagram showing expression levels of MHC class I on cancercells (human HER2-positive cells) as determined with flow cytometry andexpressed as MFI for an antibody-drug conjugate (1)-administered groupof mice with subcutaneously transplanted CT26.WT-hHER2 cells and acontrol group thereof.

FIG. 16 is a diagram showing expression levels of PD-L1 on cancer cells(human HER2-positive cells) as determined with flow cytometry andexpressed as MFI for an antibody-drug conjugate (1)-administered groupof mice with subcutaneously transplanted CT26.WT-hHER2 cells and acontrol group thereof.

FIG. 17 is a diagram showing expression levels of MHC class I asdetermined with flow cytometry for cancer cells treated with a compound(A) and those treated with DMSO.

FIG. 18 is a diagram showing transition of tumor volume for anantibody-drug conjugate (1)-administered group of mouse models of nudemice with subcutaneously transplanted CT26.WT-hHER2 cells and a controlgroup thereof.

FIG. 19 is a diagram showing transition of tumor volume for anantibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells, a control antibody-drugconjugate-administered group thereof, and a control group thereof.

FIG. 20 is a diagram showing transition of tumor volume for singleadministration groups of mice with subcutaneously transplantedEMT6-hHER2 cells with each of an antibody-drug conjugate (1) and ananti-PD-1 antibody (clone RMP1-14), and a combined administration groupthereof.

FIG. 21 is a diagram showing life-prolonging effects of different agentson mice with subcutaneously transplanted CT26.WT-hHER2 cells. Comparisonwas made on life-prolonging effect between a single administration groupwith each of an antibody-drug conjugate (1) and an anti-PD-L1 antibody(clone 10F.9G2) and a combined administration group.

FIG. 22 is a diagram showing life-prolonging effects of different agentson mice with subcutaneously transplanted EMT6-hHER2 cells. Comparisonwas made on life-prolonging effect between a single administration groupwith each of an antibody-drug conjugate (1) and an anti-PD-L1 antibody(clone 10F.9G2) and a combined administration group.

FIG. 23 is a diagram showing transition of tumor volume for singleadministration groups of mice with subcutaneously transplantedCT26.WT-hHER2 cells with each of an antibody-drug conjugate (1) and ananti-CD4 antibody, and a combined administration group thereof.

FIG. 24 is a diagram showing transition of tumor volume for singleadministration groups of mice with subcutaneously transplantedCT26.WT-hHER2 cells with each of an antibody-drug conjugate (1) and ananti-CD8 antibody, and a combined administration group thereof.

FIG. 25 is a diagram showing the fraction of CD8-positive T cells amongintratumor living cells as determined with flow cytometry for anantibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 26 is a diagram showing the fraction of Granzyme B-positive cellsamong intratumor CD8-positive T cells as determined with flow cytometryfor an antibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 27 is a diagram showing the fraction of CD8-positive T cells beingGranzyme B-positive among intratumor living cells as determined withflow cytometry for an antibody-drug conjugate (1)-administered group ofmice with subcutaneously transplanted CT26.WT-hHER2 cells and a controlgroup thereof.

FIG. 28 is a diagram showing the fraction of CD4-positive T cells amongintratumor living cells as determined with flow cytometry for anantibody-drug conjugate (1)-administered group of mice withsubcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 29 is a series of images of excised tumors stained with an anti-CD8antibody for an antibody-drug conjugate (1)-administered group of micewith subcutaneously transplanted CT26.WT-hHER2 cells and a control groupthereof.

FIG. 30 is a diagram showing the number of CD8-positive cells per unitarea in tumors for an antibody-drug conjugate (1)-administered group ofmice with subcutaneously transplanted CT26.WT-hHER2 cells and a controlgroup thereof, as counted through analysis of images of excised tumorsstained with an anti-CD8 antibody.

FIG. 31 is a diagram showing expression levels of MHC class I asdetermined with flow cytometry for cancer cells treated with a compound(A), those treated with DM1-SMe, those treated with DM4-SMe, thosetreated with MMAE, and those treated with DMSO.

FIG. 32 is a diagram showing transition of tumor volume for singleadministration groups of mice with subcutaneously transplantedEMT6-hHER2 cells with each of an antibody-drug conjugate (1) and ananti-CTLA-4 antibody (clone 9H10), and a combined administration groupthereof.

DESCRIPTION OF EMBODIMENTS

Hereinafter, preferred modes for carrying out the present invention aredescribed with reference to the drawings. The embodiments describedbelow are given merely for illustrating one example of a typicalembodiment of the present invention and are not intended to limit thescope of the present invention.

[Antibody-Drug Conjugate]

The antibody-drug conjugate used in the present invention is anantibody-drug conjugate in which a drug-linker represented by thefollowing formula:

wherein A represents the connecting position to an antibody,is conjugated to the antibody via a thioether bond.

In the present invention, the partial structure consisting of a linkerand a drug in the antibody-drug conjugate is referred to as a“drug-linker”. The drug-linker is connected to a thiol group (in otherwords, the sulfur atom of a cysteine residue) formed at an interchaindisulfide bond site (two sites between heavy chains, and two sitesbetween a heavy chain and a light chain).

The drug-linker of the present invention includes exatecan (IUPAC name:(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13-dione,(also expressed as chemical name:(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione)),which is a topoisomerase I inhibitor, as a component. Exatecan is acamptothecin derivative having an antitumor effect, represented by thefollowing formula:

The antibody-drug conjugate used in the present invention can be alsorepresented by the following formula.

Here, the drug-linker is conjugated to an antibody via a thioether bond.The meaning of n is the same as that of what is called the averagenumber of conjugated drug molecules (DAR; Drug-to-Antibody Ratio), andindicates the average number of units of the drug-linker conjugated perantibody molecule.

The antibody-drug conjugate used in the present invention has anantitumor immunity-activating effect.

In the present invention, the term “antitumor immunity-activating”refers to a property of promoting exertion of an antitumor effect byactivating at least one selected from the group consisting of T cellsand B cells (Bracci L. et al., Cell Death Differ. (2014) 21, 15-25, ChenD S. Et al., Immunity (2013) 39, 1-10, Andersen M H. et al., Journal ofInvestigative Dermatology (2006) 126, 32-41).

The situation that the antibody-drug conjugate used in the presentinvention is promoting exertion of an antitumor effect by activating atleast one selected from the group consisting of T cells and B cells canbe confirmed through comparison for the antibody-drug conjugate used inthe present invention between an antitumor effect in mice with normalimmune functions and that in mice with immune functions of T cells and Bcells impaired (nude mice).

The antibody-drug conjugate used in the present invention has at leastone effect selected from the group consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells.

The “promoting effect on growth of intratumor CD8-positive T cells”possessed by the antibody-drug conjugate used in the present inventioncan be confirmed, for example, by determining the fraction of CD45-,CD3-, CD8-positive cells (CD8-positive T cells) among living cells withflow cytometry to examine the increase rate for an antibody-drugconjugate-administered group of cancer-bearing mice and a control groupthereof. Alternatively, the effect can be confirmed by analyzing imagesof an excised tumor stained with an anti-CD8 antibody and counting thenumber of CD8-positive cells per unit area in the tumor to examine theincrease rate for an antibody-drug conjugate-administered group ofcancer-bearing mice and a control group thereof.

The “activating effect on intratumor CD8-positive T cells” possessed bythe antibody-drug conjugate used in the present invention can beconfirmed, for example, by determining the fraction of GranzymeB-positive cells among CD8-positive T cells with flow cytometry toexamine the increase rate for an antibody-drug conjugate-administeredgroup of cancer-bearing mice and a control group thereof. Alternatively,the effect can be confirmed by determining the fraction of GranzymeB-positive cells among living cells with flow cytometry to examine theincrease rate.

The antibody-drug conjugate used in the present invention has apromoting effect on the formation of immune memory against tumor. Thiseffect contributes to the above-described “antitumor immunity-activatingeffect”.

On being presented with a tumor-derived antigen from dendritic cells orcancer cells, T cells are activated to cause an immune response,exerting an antitumor effect.

In the present invention, the phrase “formation of immune memory againsttumor” refers to the phenomenon that T cells presented with atumor-derived antigen generate memory T cells therefrom and therebymemory of an immune response against the antigen is formed. Thephenomenon allows exertion of a sustained antitumor effect against tumorhaving the antigen, and further allows exertion of an antitumor effectagain on the recurrence of tumor having the antigen.

The “promoting effect on the formation of immune memory against tumor”possessed by the antibody-drug conjugate used in the present inventioncan be confirmed, for example, by administering the antibody-drugconjugate to cancer-bearing mice and retransplanting tumor to mice whichhave undergone complete tumor regression to determine the tumorproliferation (regression)-suppressing rate. Alternatively, the effectcan be confirmed by excising the spleen from each of the mice and addinga tumor-derived antigen to the spleen to determine the increase rate ofan immune response (e.g., the number of IFNγ-producing splenocytes).

The antibody-drug conjugate used in the present invention has apromoting effect on the formation of immune memory not only againsttumor expressing an antigen for the antibody in the antibody-drugconjugate, but also against tumor not expressing the antigen for theantibody in the antibody-drug conjugate in the same individual.

In the case that the antibody in the antibody-drug conjugate is ananti-HER2 antibody, for example, the antibody-drug conjugate used in thepresent invention not only has a promoting effect on the formation ofimmune memory against tumor expressing HER2, but also has a promotingeffect on the formation of immune memory against tumor not expressingHER2 in the same individual.

In addition, the antibody-drug conjugate used in the present inventionhas at least one effect selected from the group consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells.

These effects contribute to the above-described “promoting effect on theformation of immune memory against tumor”, and eventually contribute tothe above-described “antitumor immunity-activating effect”.

The “promoting effect on increase of the number of dendritic cells in atumor” possessed by the antibody-drug conjugate used in the presentinvention can be confirmed, for example, by determining the fraction ofCD11c-, MHC class II-, CD45-positive cells (dendritic cells, DCs) amongCD45-positive cells (lymphocytic cells) with flow cytometry to examinethe increase rate for an antibody-drug conjugate-administered group ofcancer-bearing mice and a control group thereof.

The “activating effect on dendritic cells” possessed by theantibody-drug conjugate used in the present invention can be confirmed,for example, by determining the fraction of dendritic cells expressingCD86 (activation marker) with flow cytometry to examine the increaserate for an antibody-drug conjugate-administered group of cancer-bearingmice and a control group thereof. Alternatively, the effect can beconfirmed by determining the expression level (MFI (mean fluorescenceintensity)) of CD86 on dendritic cells with flow cytometry to examinethe increase rate for an antibody-drug conjugate-administered group ofcancer-bearing mice and a control group thereof.

The “promoting effect on elevation of the expression level of MHC classI on cancer cells” possessed by the antibody-drug conjugate used in thepresent invention can be confirmed, for example, by determining theexpression level (MFI) of MHC class I on cancer cells with flowcytometry to examine the increase rate for an antibody-drugconjugate-administered group of cancer-bearing mice and a control groupthereof.

The antibody-drug conjugate used in the present invention occasionallyhas a promoting effect on elevation of the expression level of PD-L1 oncancer cells. Immune checkpoint inhibitors deactivate animmunosuppressive signal generated through the effect, and thereby theantibody-drug conjugate can exhibit a higher antitumor effect.Accordingly, the antibody-drug conjugate used in the present inventionis expected to exhibit a higher antitumor effect if being used incombination with an immune checkpoint inhibitor.

The “promoting effect on elevation of the expression level of PD-L1 oncancer cells” possessed by the antibody-drug conjugate used in thepresent invention can be confirmed, for example, by determining theexpression level (MFI) of PD-L1 on cancer cells with flow cytometry toexamine the increase rate for an antibody-drug conjugate-administeredgroup of cancer-bearing mice and a control group thereof.

After migrating into cancer cells, the antibody-drug conjugate used inthe present invention is cleaved at the linker portion to release acompound represented by the following formula:

(hereinafter, referred to as the “compound (A)”).

The compound (A) is inferred to be the original source of the antitumoractivity of the antibody-drug conjugate used in the present invention,and has been confirmed to have a topoisomerase I inhibitory effect(Ogitani Y. et al., Clinical Cancer Research, 2016, Oct. 15;22(20):5097-5108, Epub 2016 Mar. 29).

The compound (A) has an activating effect on dendritic cells and apromoting effect on elevation of the expression level of MHC class I oncancer cells.

The “activating effect on dendritic cells” possessed by the compound (A)can be confirmed, for example, by determining the expression level ofCD86 with flow cytometry to examine the increase rate for bonemarrow-derived dendritic cells treated with the compound (A) and thosetreated with DMSO.

The “promoting effect on elevation of the expression level of MHC classI on cancer cells” possessed by the compound (A) can be confirmed, forexample, by determining the expression level of MHC class I with flowcytometry to examine the increase rate for cancer cells treated with thecompound (A) and those treated with DMSO.

The “activating effect on dendritic cells” and “promoting effect onelevation of the expression level of MHC class I on cancer cells”possessed by the compound (A) are effects associated with the“activating effect on dendritic cells” and “promoting effect onelevation of the expression level of MHC class I on cancer cells”possessed by the antibody-drug conjugate used in the present invention.As described above, the compound (A) is a compound which is releasedfrom the antibody-drug conjugate used in the present invention after theantibody-drug conjugate used in the present invention migrates intocancer cells.

Accordingly, pharmaceutical compositions which release the compound (A)in a tumor are expected to have at least one effect selected from thegroup consisting of:

(1) a promoting effect on increase of the number of dendritic cells in atumor;

(2) an activating effect on dendritic cells; and

(3) a promoting effect on elevation of the expression level of MHC classI on cancer cells, as the antibody-drug conjugate used in the presentinvention.

Further, pharmaceutical compositions which release the compound (A) in atumor are expected to have a promoting effect on the formation of immunememory against tumor, as the antibody-drug conjugate used in the presentinvention.

As described above, the compound (A) is a compound which is generatedfrom the antibody-drug conjugate used in the present invention after theantibody-drug conjugate used in the present invention migrates intocancer cells.

Accordingly, pharmaceutical compositions which release the compound (A)in a tumor are expected to have at least one effect selected from thegroup consisting of:

(1) a promoting effect on growth of intratumor CD8-positive T cells; and

(2) an activating effect on intratumor CD8-positive T cells,

as the antibody-drug conjugate used in the present invention, and inaddition are expected to have an “antitumor immunity-activating effect”.

The antibody-drug conjugate used in the present invention is known tohave a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107,1039-1046).

The bystander effect is exerted through a process such that theantibody-drug conjugate used in the present invention is internalized incancer cells expressing a target and the compound (A) released thenexerts an antitumor effect also on cancer cells which are presenttherearound and not expressing the target.

The bystander effect possessed by the antibody-drug conjugate used inthe present invention is exerted as an excellent antitumor effect evenwhen using in combination with an immune checkpoint inhibitor.

[Antibody for Use in Production of Antibody-Drug Conjugate]

The antibody for use in production of the antibody-drug conjugateaccording to the present invention may be derived from any species, andis preferably an antibody derived from a human, a rat, a mouse, or arabbit. In cases when the antibody is derived from species other thanhuman species, it is preferably chimerized or humanized using a wellknown technique. The antibody of the present invention may be apolyclonal antibody or a monoclonal antibody and is preferably amonoclonal antibody.

The antibody for use in production of the antibody-drug conjugateaccording to the present invention is an antibody preferably having acharacteristic of being capable of targeting cancer cells, and ispreferably an antibody possessing, for example, a property ofrecognizing a cancer cell, a property of binding to a cancer cell, aproperty of internalizing in a cancer cell, and/or cytocidal activityagainst cancer cells.

The binding activity of the antibody against cancer cells can beconfirmed using flow cytometry. The internalization of the antibody intotumor cells can be confirmed using (1) an assay of visualizing anantibody incorporated in cells under a fluorescence microscope using asecondary antibody (fluorescently labeled) binding to the therapeuticantibody (Cell Death and Differentiation (2008) 15, 751-761), (2) anassay of measuring a fluorescence intensity incorporated in cells usinga secondary antibody (fluorescently labeled) binding to the therapeuticantibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December2004), or (3) a Mab-ZAP assay using an immunotoxin binding to thetherapeutic antibody wherein the toxin is released upon incorporationinto cells to inhibit cell growth (Bio Techniques 28: 162-165, January2000). As the immunotoxin, a recombinant complex protein of a diphtheriatoxin catalytic domain and protein G may be used.

The antitumor activity of the antibody can be confirmed in vitro bydetermining inhibitory activity against cell growth. For example, acancer cell line overexpressing a target protein for the antibody iscultured, and the antibody is added at varying concentrations into theculture system to determine inhibitory activity against focus formation,colony formation, and spheroid growth. The antitumor activity can beconfirmed in vivo, for example, by administering the antibody to a nudemouse with a transplanted cancer cell line highly expressing the targetprotein, and determining change in the cancer cell.

Since the compound conjugated in the antibody-drug conjugate exerts anantitumor effect, it is preferred but not essential that the antibodyitself should have an antitumor effect. For the purpose of specificallyand selectively exerting the cytotoxic activity of the antitumorcompound against cancer cells, it is important and also preferred thatthe antibody should have the property of internalizing to migrate intocancer cells.

The antibody for use in production of the antibody-drug conjugateaccording to the present invention can be obtained by a procedure knownin the art. For example, the antibody of the present invention can beobtained using a method usually carried out in the art, which involvesimmunizing animals with an antigenic polypeptide and collecting andpurifying antibodies produced in vivo. The origin of the antigen is notlimited to humans, and the animals may be immunized with an antigenderived from a non-human animal such as a mouse, a rat and the like. Inthis case, the cross-reactivity of antibodies binding to the obtainedheterologous antigen with human antigens can be tested to screen for anantibody applicable to a human disease.

Alternatively, antibody-producing cells which produce antibodies againstthe antigen are fused with myeloma cells according to a method known inthe art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; andKennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y.(1980)) to establish hybridomas, from which monoclonal antibodies can inturn be obtained.

The antigen can be obtained by genetically engineering host cells toproduce a gene encoding the antigenic protein. Specifically, vectorsthat permit expression of the antigen gene are prepared and transferredto host cells so that the gene is expressed. The antigen thus expressedcan be purified. The antibody can also be obtained by a method ofimmunizing animals with the above-described genetically engineeredantigen-expressing cells or a cell line expressing the antigen.

The antibody for use in production of the antibody-drug conjugateaccording to the present invention is preferably a recombinant antibodyobtained by artificial modification for the purpose of decreasingheterologous antigenicity to humans such as a chimeric antibody or ahumanized antibody, or is preferably an antibody having only the genesequence of an antibody derived from a human, that is, a human antibody.These antibodies can be produced using a known method.

As the chimeric antibody, an antibody in which antibody variable andconstant regions are derived from different species, for example, achimeric antibody in which a mouse- or rat-derived antibody variableregion is connected to a human-derived antibody constant region can beexemplified (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

As the humanized antibody, an antibody obtained by integrating only thecomplementarity determining region (CDR) of a heterologous antibody intoa human-derived antibody (Nature (1986) 321, pp. 522-525), and anantibody obtained by grafting a part of the amino acid residues of theframework of a heterologous antibody as well as the CDR sequence of theheterologous antibody to a human antibody by a CDR-grafting method (WO90/07861), and an antibody humanized using a gene conversion mutagenesisstrategy (U.S. Pat. No. 5,821,337) can be exemplified.

As the human antibody, an antibody generated by using a humanantibody-producing mouse having a human chromosome fragment includinggenes of a heavy chain and light chain of a human antibody (seeTomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y.et. al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. al.,Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73(Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer AcademicPublishers, 1999; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA(2000) 97, p. 722-727, etc.) can be exemplified. As an alternative, anantibody obtained by phage display, the antibody being selected from ahuman antibody library (see Wormstone, I. M. et. al, InvestigativeOphthalmology & Visual Science. (2002)43 (7), p. 2301-2308; Carmen, S.et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2),p. 189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p.427-431, etc.) can be exemplified.

In the present invention, modified variants of the antibody for use inproduction of the antibody-drug conjugate according to the presentinvention are also included. The modified variant refers to a variantobtained by subjecting the antibody according to the present inventionto chemical or biological modification. Examples of the chemicallymodified variant include variants including a linkage of a chemicalmoiety to an amino acid skeleton, variants including a linkage of achemical moiety to an N-linked or O-linked carbohydrate chain, etc.Examples of the biologically modified variant include variants obtainedby post-translational modification (such as N-linked or O-linkedglycosylation, N- or C-terminal processing, deamidation, isomerizationof aspartic acid, or oxidation of methionine), and variants in which amethionine residue has been added to the N terminus by being expressedin a prokaryotic host cell. Further, an antibody labeled so as to enablethe detection or isolation of the antibody or an antigen according tothe present invention, for example, an enzyme-labeled antibody, afluorescence-labeled antibody, and an affinity-labeled antibody are alsoincluded in the meaning of the modified variant. Such a modified variantof the antibody according to the present invention is useful forimproving the stability and blood retention of the antibody, reducingthe antigenicity thereof, detecting or isolating an antibody or anantigen, and so on.

Further, by regulating the modification of a glycan which is linked tothe antibody according to the present invention (glycosylation,defucosylation, etc.), it is possible to enhance antibody-dependentcellular cytotoxic activity. As the technique for regulating themodification of a glycan of antibodies, WO 99/54342, WO 00/61739, WO02/31140, etc. are known. However, the technique is not limited thereto.In the antibody according to the present invention, antibodies in whichthe modification of a glycan is regulated are also included.

It is known that a lysine residue at the carboxyl terminus of the heavychain of an antibody produced in a cultured mammalian cell is deleted(Journal of Chromatography A, 705: 129-134 (1995)), and it is also knownthat two amino acid residues (glycine and lysine) at the carboxylterminus of the heavy chain of an antibody produced in a culturedmammalian cell are deleted and a proline residue newly located at thecarboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83(2007)). However, such deletion and modification of the heavy chainsequence do not affect the antigen-binding affinity and the effectorfunction (the activation of complement, antibody-dependent cellularcytotoxicity, etc.) of the antibody. Therefore, in the antibodyaccording to the present invention, antibodies subjected to suchmodification and functional fragments of the antibody are also included,and deletion variants in which one or two amino acids have been deletedat the carboxyl terminus of the heavy chain, variants obtained byamidation of deletion variants (for example, a heavy chain in which thecarboxyl terminal proline residue has been amidated), and the like arealso included. The type of deletion variant having a deletion at thecarboxyl terminus of the heavy chain of the antibody according to thepresent invention is not limited to the above variants as long as theantigen-binding affinity and the effector function are conserved. Thetwo heavy chains constituting the antibody according to the presentinvention may be of one type selected from the group consisting of afull-length heavy chain and the above-described deletion variant, or maybe of two types in combination selected therefrom. The ratio of theamount of each deletion variant can be affected by the type of culturedmammalian cells which produce the antibody according to the presentinvention and the culture conditions; however, an antibody in which oneamino acid residue at the carboxyl terminus has been deleted in both ofthe two heavy chains in the antibody according to the present inventioncan be preferably exemplified.

As isotypes of the antibody according to the present invention, forexample, IgG (IgG1, IgG2, IgG3, IgG4) can be exemplified, and IgG1 orIgG2 can be exemplified preferably.

Examples of antibodies applicable to production of the antibody-drugconjugate according to the present invention can include, but are notparticularly limited to, an anti-HER2 antibody, an anti-HER3 antibody,an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-CD3 antibody, ananti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, ananti-CD56 antibody, an anti-CD98 antibody, an anti-DR5 antibody, ananti-EGFR antibody, an anti-EPHA2 antibody, an anti-FGFR2 antibody, ananti-FGFR4 antibody, an anti-FOLR1 antibody, an anti-VEGF antibody, ananti-CD20 antibody, an anti-CD22 antibody, an anti-CD70 antibody, ananti-PSMA antibody, an anti-CEA antibody, and an anti-Mesothelinantibody, and an anti-HER2 antibody, an anti-HER3 antibody, ananti-TROP2 antibody, and an anti-B7-H3 antibody can be preferablyexemplified, and an anti-HER2 antibody can be more preferablyexemplified.

In the present invention, the term “anti-HER2 antibody” refers to anantibody which specifically binds to HER2 (Human Epidermal Growth FactorReceptor Type 2; ErbB-2), and preferably has an activity ofinternalizing in HER2-expressing cells by binding to HER2.

Examples of the anti-HER2 antibody include trastuzumab (U.S. Pat. No.5,821,337) and pertuzumab (International Publication No. WO 01/00245),and trastuzumab can be preferably exemplified.

In the present invention, the term “trastuzumab” is also calledHERCEPTIN (registered trademark), huMAb4D5-8, or rhuMAb4D5-8 and is ahumanized anti-HER2 antibody comprising a heavy chain consisting of anamino acid sequence consisting of amino acid residues 1 to 449 of SEQ IDNO: 1 (FIG. 1) and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 1 to 214 of SEQ ID NO: 2 (FIG. 2).

A preferred anti-HER2 antibody for use in production of theantibody-drug conjugate according to the present invention is:

(1) an antibody comprising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 anda light chain consisting of an amino acid sequence consisting of aminoacid residues 1 to 214 of SEQ ID NO: 2; or

(2) an antibody comprising a heavy chain consisting of the amino acidsequence represented by SEQ ID NO: 1 and a light chain consisting of theamino acid sequence represented by SEQ ID NO: 2.

In the present invention, the term “anti-HER3 antibody” refers to anantibody which specifically binds to HER3 (Human Epidermal Growth FactorReceptor Type 3; ErbB-3), and preferably has an activity ofinternalizing in HER3-expressing cells by binding to HER3.

Examples of the anti-HER3 antibody include patritumab (U3-1287), U1-59(International Publication No. WO 2007/077028), MM-121 (seribantumab),an anti-ERBB3 antibody described in International Publication No. WO2008/100624, RG-7116 (lumretuzumab), and LJM-716 (elgemtumab), andpatritumab and U1-59 can be preferably exemplified.

In the present invention, the term “anti-TROP2 antibody” refers to anantibody which specifically binds to TROP2 (TACSTD2: Tumor-associatedcalcium signal transducer 2; EGP-1), and preferably has an activity ofinternalizing in TROP2-expressing cells by binding to TROP2.

Examples of the anti-TROP2 antibody include hTINA1-H1L1 (InternationalPublication No. WO 2015/098099).

In the present invention, the term “anti-B7-H3 antibody” refers to anantibody which specifically binds to B7-H3, and preferably has anactivity of internalizing in B7-H3-expressing cells by binding to B7-H3.

Examples of the anti-B7-H3 antibody include M30-H1-L4 (InternationalPublication No. WO 2014/057687).

[Drug-Linker Intermediate for Use in Production of Antibody-DrugConjugate]

A drug-linker intermediate for use in production of the antibody-drugconjugate according to the present invention is represented by thefollowing formula.

The drug-linker intermediate can be expressed as the chemical nameN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide,and can be produced with reference to descriptions in InternationalPublication No. WO 2014/057687, International Publication No. WO2015/098099, International Publication No. WO 2015/115091, InternationalPublication No. WO 2015/155998, and so on.

[Conjugation Between Antibody and Drug-Linker Intermediate]

The antibody-drug conjugate used in the present invention can beproduced by reacting the above-described drug-linker intermediate and anantibody having a thiol group (or referred to as a sulfhydryl group).

The antibody having a sulfhydryl group can be obtained by a method wellknown in the art (Hermanson, G. T, Bioconjugate Techniques, pp. 56-136,pp. 456-493, Academic Press (1996)). For example, by using 0.3 to 3molar equivalents of a reducing agent such astris(2-carboxyethyl)phosphine hydrochloride (TCEP) per interchaindisulfide within the antibody and reacting with the antibody in a buffersolution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an antibody having a sulfhydryl group withpartially or completely reduced interchain disulfides within theantibody can be obtained.

Further, by using 2 to 20 molar equivalents of the drug-linkerintermediate per the antibody having a sulfhydryl group, anantibody-drug conjugate in which 2 to 8 drug molecules are conjugatedper antibody molecule can be produced.

The average number of conjugated drug molecules per antibody molecule ofthe antibody-drug conjugate produced can be determined, for example, bya method of calculation based on measurement of UV absorbance for theantibody-drug conjugate and the conjugation precursor thereof at twowavelengths of 280 nm and 370 nm (UV method), or a method of calculationbased on quantification through HPLC measurement for fragments obtainedby treating the antibody-drug conjugate with a reducing agent (HPLCmethod).

Conjugation between the antibody and the drug-linker intermediate andcalculation of the average number of conjugated drug molecules perantibody molecule of the antibody-drug conjugate can be performed withreference to descriptions in International Publication No. WO2014/057687, International Publication No. WO 2015/098099, InternationalPublication No. WO 2015/115091, International Publication No. WO2015/155998, and so on.

In the present invention, the term “anti-HER2 antibody-drug conjugate”refers to an antibody-drug conjugate such that the antibody in anantibody-drug conjugate is an anti-HER2 antibody.

The average number of units of the drug-linker conjugated per antibodymolecule in the anti-HER2 antibody-drug conjugate used in the presentinvention is preferably 2 to 8, more preferably 3 to 8, even morepreferably 7 to 8, even more preferably 7.5 to 8, and even morepreferably about 8.

The anti-HER2 antibody-drug conjugate used in the present invention canbe produced with reference to descriptions in International PublicationNo. WO 2015/115091 and so on.

In the present invention, the term “anti-HER3 antibody-drug conjugate”refers to an antibody-drug conjugate such that the antibody in anantibody-drug conjugate is an anti-HER3 antibody.

The average number of units of the drug-linker conjugated per antibodymolecule in the anti-HER3 antibody-drug conjugate used in the presentinvention is preferably 2 to 8, more preferably 3 to 8, even morepreferably 7 to 8, even more preferably 7.5 to 8, and even morepreferably about 8.

The anti-HER3 antibody-drug conjugate used in the present invention canbe produced with reference to descriptions in International PublicationNo. WO 2015/155998 and so on.

In the present invention, the term “anti-TROP2 antibody-drug conjugate”refers to an antibody-drug conjugate such that the antibody in anantibody-drug conjugate is an anti-TROP2 antibody.

The average number of units of the drug-linker conjugated per antibodymolecule in the anti-TROP2 antibody-drug conjugate used in the presentinvention is preferably 2 to 8, more preferably 3 to 5, even morepreferably 3.5 to 4.5, and even more preferably about 4.

The anti-TROP2 antibody-drug conjugate used in the present invention canbe produced with reference to descriptions in International PublicationNo. WO 2015/098099 and so on.

In the present invention, the term “anti-B7-H3 antibody-drug conjugate”refers to an antibody-drug conjugate such that the antibody in anantibody-drug conjugate is an anti-B7-H3 antibody.

The average number of units of the drug-linker conjugated per antibodymolecule in the anti-B7-H3 antibody-drug conjugate used in the presentinvention is preferably 2 to 8, more preferably 3 to 5, even morepreferably 3.5 to 4.5, and even more preferably about 4.

The anti-B7-H3 antibody-drug conjugate used in the present invention canbe produced with reference to descriptions in International PublicationNo. WO 2014/057687 and so on.

[Immune Checkpoint Inhibitor]

In the present invention, the term “immune checkpoint inhibitor” refersto an agent which inhibits the immune suppression system to activatetumor immunity.

Preferred examples of the immune checkpoint inhibitor used in thepresent invention can include, but not particularly limited to, ananti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody,and an anti-PD-1 antibody and an anti-PD-L1 antibody can be morepreferably exemplified.

In the present invention, the term “anti-PD-1 antibody” refers to anantibody which specifically binds to PD-1 (Programmed cell death-1;CD279; PDCD1), and has an activity of reducing, inhibiting, and/orinterfering with signal transduction caused by interaction between PD-1and PD-L1 or PD-L2 as a binding partner. The anti-PD-1 antibody used inthe present invention is not particularly limited as long as theclinical efficacy and safety thereof have been confirmed, and nivolumab(International Publication No. WO 2006/121168, etc.) and pembrolizumab(International Publication No. WO 2008/156712, etc.) can be preferablyexemplified. For the purpose of confirming the effect of use incombination with the antibody-drug conjugate used in the presentinvention in a preclinical study, a commercially available anti-PD-1antibody for research (e.g., clone RMP1-14) and so on can be used.

In the present invention, the term “anti-PD-L1 antibody” refers to anantibody which specifically binds to PD-L1 (Programmed cell death ligand1; CD274; B7-H1), and has an activity of reducing, inhibiting, and/orinterfering with signal transduction caused by interaction between PD-L1and PD-1 or B7.1 (CD80) as a binding partner. The anti-PD-L1 antibodyused in the present invention is not particularly limited as long as theclinical efficacy and safety thereof have been confirmed, andatezolizumab (International Publication No. WO 2010/077634, etc.),durvalumab (International Publication No. WO 2011/066389, etc.), andavelumab (International Publication No. WO 2013/079174, etc.) can bepreferably exemplified. For the purpose of confirming the effect of usein combination with the antibody-drug conjugate used in the presentinvention in a preclinical study, a commercially available anti-PD-L1antibody for research (e.g., clone 10F.9G2) and so on can be used.

In the present invention, the term “anti-CTLA-4 antibody” refers to anantibody which specifically binds to CTLA-4 (CytotoxicT-lymphocyte-associated protein 4; CD152), and has an activity ofreducing, inhibiting, and/or interfering with signal transduction causedby interaction between CTLA-4 and B7.1 (CD80) or B7.2 (CD86) as abinding partner. The anti-CTLA-4 antibody used in the present inventionis not particularly limited as long as the clinical efficacy and safetythereof have been confirmed, and ipilimumab (International PublicationNo. WO 2001/014424, etc.) and tremelimumab (International PublicationNo. WO 2000/037504, etc.) can be preferably exemplified. For the purposeof confirming the effect of use in combination with the antibody-drugconjugate used in the present invention in a preclinical study, acommercially available anti-CTLA-4 antibody for research (e.g., clone9H10) and so on can be used.

[Medicines]

Described in the following are a pharmaceutical composition and atherapeutic method wherein the antibody-drug conjugate according to thepresent invention and an immune checkpoint inhibitor are administered incombination, and a pharmaceutical composition and a therapeutic methodfor use in treatment of a disease that can be ameliorated through anantitumor immunity-activating effect wherein the antibody-drug conjugateaccording to the present invention is included.

The pharmaceutical composition and therapeutic method of the presentinvention may be characterized in that the antibody-drug conjugate andthe immune checkpoint inhibitor are separately contained as activecomponents in different formulations, and are administeredsimultaneously or at different times, or characterized in that theantibody-drug conjugate and the immune checkpoint inhibitor arecontained as active components in a single formulation and administered.The pharmaceutical composition and therapeutic method according to thepresent invention may be such that the antibody-drug conjugate accordingto the present invention is contained as an active component in a singleformulation and administered for treating a disease that can beameliorated through an antitumor immunity-activating effect.

The pharmaceutical composition and therapeutic method of the presentinvention can be used for treating cancer, and can be preferably usedfor treating at least one disease selected from the group consisting oflung cancer (including non-small cell lung cancer), urothelial cancer,colorectal cancer (also called colon and rectal cancer, and includingcolon cancer and rectal cancer), prostate cancer, ovarian cancer,pancreatic cancer, breast cancer, bladder cancer, gastric cancer (alsocalled gastric adenocarcinoma), esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.

The pharmaceutical composition and therapeutic method of the presentinvention can be selectively used as an agent for drug therapy, which isa main method for treating cancer, and as a result, can delaydevelopment of cancer cells, inhibit growth thereof, and further killcancer cells. These effects can allow cancer patients to be free fromsymptoms caused by cancer or achieve improvement in QOL of cancerpatients and attain a therapeutic effect by sustaining the lives of thecancer patients. Even if the pharmaceutical composition and therapeuticmethod of the present invention do not accomplish killing cancer cells,they can achieve higher QOL of cancer patients while achievinglonger-term survival, by inhibiting or controlling the growth of cancercells.

In such drug therapy, the pharmaceutical composition and therapeuticmethod of the present invention can be used as an agent alone and inaddition, they can be used as an agent in combination with an additionaltherapy in adjuvant therapy and can be combined with surgical operation,radiotherapy, hormone therapy, or the like. Furthermore, they can alsobe used as an agent for drug therapy in neoadjuvant therapy.

In addition to the therapeutic use as described above, for example, aprophylactic effect such as suppressing the growth of small metastaticcancer cells and further killing them can also be expected for thepharmaceutical composition and therapeutic method according to thepresent invention. For example, an effect of inhibiting and killingcancer cells in a body fluid in the course of metastasis or an effectof, for example, inhibiting and killing small cancer cells immediatelyafter implantation in any tissue can be expected. Accordingly,inhibition of cancer metastasis or a prophylactic effect can beexpected, particularly, after surgical removal of cancer.

The pharmaceutical composition and therapeutic method of the presentinvention can be expected to exert a therapeutic effect by applicationas systemic therapy to patients, and additionally, by local applicationto cancer tissues.

The pharmaceutical composition and therapeutic method of the presentinvention can be preferably used for a mammal, but are more preferablyused for a human.

The pharmaceutical composition of the present invention can beadministered as a pharmaceutical composition containing at least onepharmaceutically suitable ingredient. Substances used in thepharmaceutical composition of the present invention can be suitablyselected and applied from formulation additives or the like that aregenerally used in the art, in view of the dosage or administrationconcentration. For example, the pharmaceutical composition abovetypically contains at least one pharmaceutical carrier (for example,sterilized liquid). Herein, the liquid includes, for example, water andoil (petroleum oil and oil of animal origin, plant origin, or syntheticorigin). The oil may be, for example, peanut oil, soybean oil, mineraloil, or sesame oil. Water is a more typical carrier when thepharmaceutical composition above is intravenously administered. Salinesolution, an aqueous dextrose solution, and an aqueous glycerol solutioncan be also used as a liquid carrier, in particular, for an injectionsolution. A suitable pharmaceutical vehicle can be selected from onesknown in the art. If desired, the composition above may also contain atrace amount of a moisturizing agent, an emulsifying agent, or a pHbuffering agent. Examples of suitable pharmaceutical carriers aredisclosed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulations correspond to the administration mode.

Various delivery systems are known and they can be used foradministering the pharmaceutical composition of the present invention.Examples of the administration route can include intradermal,intramuscular, intraperitoneal, intravenous, and subcutaneous routes,but are not limited thereto. The administration can be made by injectionor bolus injection, for example. According to a specific preferredembodiment, the administration of the antibody-drug conjugate and immunecheckpoint inhibitor used in the present invention is performed byinjection. Parenteral administration is a preferred administrationroute.

According to a representative embodiment, the pharmaceutical compositionis prescribed, as a pharmaceutical composition suitable for intravenousadministration to humans, according to conventional procedures. Thecomposition for intravenous administration is typically a solution in asterile and isotonic aqueous buffer solution. If necessary, thepharmaceutical composition may contain a solubilizing agent and localanesthetics to alleviate pain at an injection site (for example,lignocaine). Generally, the ingredient above is provided individually asany one of a lyophilized powder or an anhydrous concentrate contained ina container which is obtained by sealing in an ampoule or a sachethaving an amount of the active agent or as a mixture in a unit dosageform. When the pharmaceutical composition is to be administered byinjection, it may be administered from an injection bottle containingwater or saline of sterile pharmaceutical grade. When the pharmaceuticalcomposition is administered by injection, an ampoule of sterile water orsaline for injection may be provided such that the aforementionedingredients are admixed with each other before administration.

The pharmaceutical composition and therapeutic method of the presentinvention may include a cancer treating agent other than theantibody-drug conjugate and immune checkpoint inhibitor according to thepresent invention. The pharmaceutical composition and therapeutic methodof the present invention can be administered in combination with othercancer treating agents. The anti-cancer effect may be enhancedaccordingly. Other anti-cancer agents used for such purpose may beadministered to an individual simultaneously with, separately from, orsubsequently to the pharmaceutical composition of the present invention,and may be administered while varying the administration interval foreach. Examples of cancer treating agents include 5-fluorouracil (5-FU),pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin,gemcitabine, capecitabine, irinotecan (CPT-11), docetaxel, pemetrexed,sorafenib, vinblastin, vinorelbine, everolims, tanespimycin,bevacizumab, oxaliplatin, lapatinib, trastuzumab emtansine (T-DM1) oragents described in International Publication No. WO 2003/038043, LH-RHalagogues (leuprorelin, goserelin, or the like), estramustine phosphate,estrogen antagonists (tamoxifen, raloxifene, or the like), and aromataseinhibitors (anastrozole, letrozole, exemestane, or the like), but arenot limited as long as they are agents having an antitumor activity.

The pharmaceutical composition can be formulated into a lyophilizationformulation or a liquid formulation as a formulation having the desiredcomposition and required purity. When formulated as a lyophilizationformulation, it may be a formulation containing suitable formulationadditives that are used in the art. Also for a liquid formulation, itcan be formulated as a liquid formulation containing various formulationadditives that are used in the art.

The composition and concentration of the pharmaceutical composition mayvary depending on the administration method. However, the antibody-drugconjugate and immune checkpoint inhibitor contained in thepharmaceutical composition of the present invention can exhibit apharmaceutical effect even at a small dosage when the antibody-drugconjugate has a higher affinity for an antigen, that is, a higheraffinity (=lower Kd value) in terms of the dissociation constant (thatis, Kd value) for the antigen. Thus, for determining the dosage of theantibody-drug conjugate and immune checkpoint inhibitor, the dosage canbe determined in view of the situation relating to the affinity with theantigen. When the antibody-drug conjugate and immune checkpointinhibitor according to the present invention are administered to ahuman, for example, about 0.001 to 100 mg/kg can be administered once oradministered in several portions with intervals of 1 to 180 days.

Examples of administration methods for the antibody-drug conjugateaccording to the present invention include a method of administering 0.8mg/kg to 8 mg/kg once every three weeks. Examples of the dose include0.8 mg/kg, 1.6 mg/kg, 3.2 mg/kg, 5.4 mg/kg, 6.4 mg/kg, 7.4 mg/kg, and 8mg/kg. Although it is sufficient to administer once every three weeks(q3w), administration may be performed once a week (q1w), once every twoweeks (q2w), or once every four weeks (q4w).

EXAMPLES

The present invention is specifically described in view of the examplesshown below. However, the present invention is not limited to these.Further, it is by no means to be interpreted in a limited way.

Production Example 1: Preparation of Antibody-Drug Conjugate

In accordance with a production method described in InternationalPublication No. WO 2015/115091 with use of a humanized anti-HER2antibody (trastuzumab), an antibody-drug conjugate in which adrug-linker represented by the following formula:

wherein A represents the connecting position to an antibody,is conjugated to the anti-HER2 antibody via a thioether bond(hereinafter, referred to as “the antibody-drug conjugate (1)”) wasproduced.

Production Example 2: Preparation of Compound (A)

In accordance with a production method described in InternationalPublication No. WO 2014/057687, a compound represented by the followingformula:

(compound (A)) was produced.

Evaluation Example 1: Life Prolongation Test

Mouse: 6-week-old female BALB/c mice (BALB/c AnNCrlCrlj) (Charles RiverLaboratories Japan, Inc.) were subjected to experiment.

Assay and calculation expression: The major axis and minor axis of atumor were measured twice a week by using an electronic digital caliper(CD15-CX, Mitutoyo Corp.), and the tumor volume (mm³) was calculated.The calculation expression is as shown below.Tumor volume (mm³)=0.5×Major axis (mm)×[Minor axis (mm)]²

From the viewpoint of animal testing ethics, individuals whose tumorvolume exceeded 3000 mm³ were euthanized.

The antibody-drug conjugate (1) (Drug-to-Antibody Ratio: 7.6) wasdiluted with special solvent (10 mM Histidine, 10% Trehalose, 0.02%Polysorbate 20, pH 5.5) for use. An anti-PD-1 antibody (clone RMP1-14)was purchased from Bio X Cell, and diluted with DPBS (Sigma-Aldrich Co.LLC) for use. In administration, a dose of 10 mL/kg was intravenouslyadministered to the tail vein of each mouse.

A human HER2 gene was transfected into the mouse colorectal cancer cellline CT26.WT (CRL2638) purchased from American Type Culture Collectionwith a retrovirus vector to prepare CT26.WT-hHER2 cells for use. Thesecells were expressing human HER2 protein on their cell membranes. TheCT26.WT-hHER2 cells were suspended in physiological saline, and 5.0×10⁶cells were subcutaneously transplanted to the right axilla of eachBALB/c mouse, and the mice were randomly grouped 6 days thereafter (Day0). The antibody-drug conjugate (1) was intravenously administered tothe tail vein of each mouse at a dose of 10 mg/kg on Days 0 and 7, twicein total. The anti-PD-1 antibody was intravenously administered to thetail vein of each mouse at a dose of 2.5 mg/kg on Days 0, 3, 7, 10, and14, five times in total. A combined administration group with theantibody-drug conjugate (1) and the anti-PD-1 antibody was established,and a group with administration of the special solvent for theantibody-drug conjugate (1) was established as a control group. Thenumber of mice in each group was six, and tumor volumes were measureduntil Day 43.

The results are shown in FIG. 3. Kaplan-Meier curves are shown therein,where the timing when tumor volume exceeded 3000 mm³ was regarded as theend point. The ordinate depicts survival rates (%) and the abscissadepicts days from the day of initial administration. For the controlgroup, drop-out was found from Day 17, and all of the mice weredetermined as subjects of euthanasia by Day 24. For the antibody-drugconjugate (1) group, in contrast, drop-out was found from Day 28, andthree mice survived until Day 43. For the anti-PD-1 antibody group,drop-out was found from Day 21, and two mice survived until Day 43.Moreover, for the combined administration group with these two agents,all the mice survived until Day 43. Weight loss was observed for none ofthe mice in all of the groups in this test. From the results, theantitumor effect of single administration of each agent was confirmed,and it was further confirmed that such effect is dramatically enhancedthrough use of the two agents in combination.

Evaluation Example 2: Life Prolongation Test

A test was conducted in the same manner as in Evaluation Example 1. Theanti-PD-1 antibody was intravenously administered to the tail vein ofeach mouse at a dose of 5 mg/kg on Days 0, 3, 7, and 10, four times intotal, where the number of mice in each group was 20, and tumor volumeswere measured until Day 38. Comparison on pharmaceutical effect betweenthe control group and each of the antibody-drug conjugate (1) group andthe anti-PD-1 antibody group, and comparison on pharmaceutical effectbetween each of the antibody-drug conjugate (1) group and the anti-PD-1antibody group and the combined administration group with both agentswere performed by using the Kaplan-Meier method/logrank test (comparisonamong multiple groups). The day when estimated tumor volume exceeded3000 mm³ (day of euthanasia) was defined as the day of event occurrence(day of death). P values adjusted for multiplicity were expressed asnumerical values to the fourth decimal place, and values of P<0.05(two-tailed test) were regarded as significant differences.

The results are shown in FIG. 4. The antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the control group(P=0.0001). The anti-PD-1 antibody group exhibited a significantlysuperior antitumor effect to the control group (P=0.0010). Further, thecombined administration group exhibited a significantly superiorantitumor effect to the antibody-drug conjugate (1) group (P=0.0006).The combined administration group exhibited a significantly superiorantitumor effect to the anti-PD-1 antibody group (P<0.0001).

Evaluation Example 3: Retransplantation Test

The antibody-drug conjugate (1) was administered to mice withsubcutaneously transplanted CT26.WT-hHER2 cells in the same manner as inEvaluation Example 1. The mice were randomly grouped 5 days after thetransplantation. From these mice, mice whose tumor completelydisappeared were selected (hereinafter, referred to as “antibody-drugconjugate (1)-treated cured mice”). Untreated mice were used for acontrol (hereinafter, referred to as “control mice”).

Subsequently, 5.0×10⁶ cells of CT26.WT-hHER2 cells or CT26.WT-mock cellswere subcutaneously transplanted to the left axilla of each of theantibody-drug conjugate (1)-treated cured mice and the control mice(retransplantation, Day 0), and tumor volumes were measured until Day17. The number of mice in each group was nine.

The results are shown in FIG. 5. The ordinate depicts tumor volumes(mm³) and the abscissa depicts days from the day of retransplantation.Tumor growth was found for the control mice with retransplantedCT26.WT-hHER2 cells and those with retransplanted CT26.WT-mock cells. Incontrast, almost no tumor growth, thus, tumor rejection was found forthe antibody-drug conjugate (1)-treated cured mice with retransplantedCT26.WT-hHER2 cells and those with retransplanted CT26.WT-mock cells.From these results, administration of the antibody-drug conjugate (1)was confirmed to cause the formation of immune memory against the tumor.

Evaluation Example 4: ELISPOT Analysis

This analysis was performed by using Murine IFNγ Single-Color EnzymaticELISPOT Assay. The spleen was excised from each of the mice used inEvaluation Example 3, and 1.0×10⁶ cells/mL of splenocytes were preparedtherefrom with CTL test medium. CT26.WT-hHER2 cells and CT26.WT-mockcells were each treated with 10 μg/mL of mitomycin C for 2 hours andwashed, and the cells were then collected, and 1.0×10⁶ cells/mL of cellswere prepared with CTL test medium, which was used as an antigen. Thesplenocytes and the antigen were added to an anti-IFNγ antibody-coatedPVDF-membrane plate each at 100 μL/well, and co-cultured at 37° C. for24 hours, and then the number of IFNγ-producing splenocytes was counted.Comparison between the control group and the antibody-drug conjugate (1)group was performed by using the Wilcoxon rank sum test, P values wereexpressed as numerical values to the fourth decimal place, and values ofP<0.05 (two-tailed test) were regarded as significant differences.

The results are shown in FIGS. 6 to 9. The CT26.WT-hHER2 cell-derivedantigen was found to give a significantly larger number ofIFNγ-producing splenocytes for the splenocytes of the antibody-drugconjugate (1)-treated cured mice with retransplanted CT26.WT-hHER2 cellsthan for the splenocytes of the control mice (P=0.0012, FIG. 6). TheCT26.WT-mock cell-derived antigen was also found to give a significantlylarger number of IFNγ-producing splenocytes (P=0.0008, FIG. 7).

Further, even in the cases involving retransplantation of CT26.WT-mockcells, the CT26.WT-hHER2 cell-derived antigen was found to give asignificantly larger number of IFNγ-producing splenocytes for thesplenocytes of the antibody-drug conjugate (1)-treated cured mice thanfor the splenocytes of the control mice (P=0.0116, FIG. 8). TheCT26.WT-mock cell-derived antigen was also found to give a significantlylarger number of IFNγ-producing splenocytes (P=0.0052, FIG. 9).

These results suggested that T cells which recognize a CT26.WTcell-derived antigen other than human HER2 had been induced in theantibody-drug conjugate (1)-treated cured mice.

The results of Evaluation Examples 3 and 4 demonstrated that theantibody-drug conjugate (1) had a promoting effect on the formation ofimmune memory against tumor. The effect was found not only for tumorexpressing HER2 but also for tumor derived from the same origin and notexpressing HER2.

Thus, it was revealed that the antibody-drug conjugate used in thepresent invention has a promoting effect on the formation of immunememory, not only against tumor expressing the antigen for the antibodyin the antibody-drug conjugate, but also against tumor not expressingthe antigen for the antibody in the antibody-drug conjugate in the sameindividual.

Evaluation Example 5: Evaluation of Effect on In Vitro Dendritic Cells

BALB/c mice were euthanized, and bone marrow cells were then separatedfrom each femur, and cultured with an RPMI 1640 medium containing 10%FBS, 55 μM 2-mercaptoethanol, 100 U/mL penicillin, 100 U/mLstreptomycin, 1 mM sodium pyruvate, 1×non-essential amino acid, 2 mML-glutamine, and 10 ng/mL mouse GM-CSF for 11 days to induce bonemarrow-derived dendritic cells. To the culture solution for the induceddendritic cells, the compound (A) was added to a concentration of 0.0625μM, 0.125 μM, 0.25 μM, 0.5 μM, or 1 μM. For a control, DMSO in aquantity equal to that of the compound (A) was added. After 24 hours,staining was performed by using a Pacific Blue labeled anti-mouse CD45Antibody (103126, BioLegend), PE labeled anti-mouse CD86 (B7-2) (553692,Becton Dickinson), APC labeled anti-mouse CD11c (550261, BectonDickinson), and FITC labeled anti-mouse MHC Class II (I-A/I-E)(11-5321-85, Thermo Fisher Scientific), and analysis was performed byusing an FACS Canto II. Dead cells had been stained with a LIVE/DEADFixable Near-IR Dead Cell Stain Kit purchased from Thermo FisherScientific, and excluded from the analysis.

FIGS. 10 and 11 show measurement results of flow cytometry forCD11c-positive cells in terms of expression levels of CD86 and MHC classII, respectively. It was found that treatment with the compound (A)elevated expression levels of both CD86 and MHC class II, which aremature/activation markers for dendritic cells, as compared with the casewith DMSO as the control.

The results of Evaluation Example 5 demonstrated that the compound (A)has an activating effect on dendritic cells.

Evaluation Example 6: Analysis of Intratumor Dendritic Cells

CT26.WT-hHER2 cells were transplanted to mice in the same manner as inEvaluation Example 1, and the mice were randomly grouped 8 daysthereafter (Day 0). The antibody-drug conjugate (1) was intravenouslyadministered to the tail vein of each mouse at a dose of 10 mg/kg on Day0. A group with administration of the special solvent for theantibody-drug conjugate (1) was established as a control group. Thenumber of mice in each group was seven. The mice were euthanized on Day8, and tumors were excised. Single cell suspensions were prepared fromthe tumors by using a Tumor Dissociation Kit, mouse, purchased fromMiltenyi Biotec, and stained and analyzed in the same manner as inEvaluation Example 5. Comparison between the control group and theantibody-drug conjugate (1) group was performed by using Student'st-test, P values were expressed as numerical values to the fourthdecimal place, and values of P<0.05 (two-tailed test) were regarded assignificant differences.

The results are shown in FIGS. 12 to 14.

It was found that the fraction of CD11c-, MHC class II-, CD45-positivecells (dendritic cells, DC) among CD45-positive cells (lymphocyticcells) in tumors significantly increased by administration of theantibody-drug conjugate (1) (FIG. 12).

Further, it was found that the number of dendritic cells expressing CD86(activation marker) significantly increased by administration of theantibody-drug conjugate (1) (FIG. 13).

Furthermore, it was found that the expression level of CD86 on dendriticcells determined in terms of MFI (mean fluorescence intensity) wassignificantly elevated by administration of the antibody-drug conjugate(1) (FIG. 14).

These results confirmed that administration of the antibody-drugconjugate (1) to cancer-bearing mice results in increase of the numberof dendritic cells among intratumor lymphocytes, increase of the numberof CD86-positive cells among intratumor dendritic cells, and elevationof the expression level of CD86 on dendritic cells.

It has been demonstrated from the results of Evaluation Example 5 thatthe compound (A), which is a drug released from the antibody-drugconjugate (1), itself has an activating effect on dendritic cells. The“activating effect on dendritic cells” possessed by the compound (A) isan effect associated with the “activating effect on dendritic cells”possessed by the antibody-drug conjugate used in the present invention.The compound (A) is a compound which is generated from the antibody-drugconjugate used in the present invention after the antibody-drugconjugate used in the present invention migrates into cancer cells.Accordingly, the compound (A) is expected to have the same effect evenin an antibody-drug conjugate in which the antibody portion is not ananti-HER2 antibody.

Evaluation Example 7: Analysis of Intratumor Cancer Cells

Cell suspensions were prepared in the same manner as in EvaluationExample 6, and then staining was performed with PE labeled anti-humanHer2/neu (340552, Becton Dickinson), APC labeled anti-mouse CD274(B7-H1, PD-L1) (124312, BioLegend), and FITC labeled anti-mouse H-2Dd(110606, BioLegend), and expression levels of MHC class I and expressionlevels of PD-L1 on cancer cells were determined with flow cytometry.Dead cells had been stained with a LIVE/DEAD Fixable Near-IR Dead CellStain Kit purchased from Thermo Fisher Scientific, and excluded from theanalysis. Comparison between the control group and the antibody-drugconjugate (1) group was performed by using Student's t-test, P valueswere expressed as numerical values to the fourth decimal place, andvalues of P<0.05 (two-tailed test) were regarded as significantdifferences.

The results are shown in FIGS. 15 and 16.

It was found that the expression level of MHC class I on cancer cells(human HER2-positive cells) was significantly elevated by administrationof the antibody-drug conjugate (1) (FIG. 15). MHC class I is a moleculenecessary when T cells recognize cancer cells. Hence, it was suggestedthat the antibody-drug conjugate (1) activates antitumor immunitythrough promoting elevation of the expression level of MHC class I oncancer cells.

It was further found that the expression level of PD-L1 on cancer cellswas significantly elevated by the antibody-drug conjugate (1) (FIG. 16).PD-L1 is known to act on PD-1 on T cells to elicit an immunosuppressivesignal. Hence, it was suggested that the antibody-drug conjugate (1)activates antitumor immunity through promoting elevation of theexpression level of PD-L1 on cancer cells, and combined use with a PD-1antibody is expected to deactivate the suppressive signal, resulting ina higher antitumor effect.

Evaluation Example 8: Analysis of In Vitro Cancer Cells

To culture solution for CT26.WT-hHER2 cells, the compound (A) was addedto a concentration of 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, or 1 μM. Fora control, DMSO in a quantity equal to that of the compound (A) wasadded. After 24 hours, staining was performed by using PE labeledanti-human Her2/neu (340552, Becton Dickinson) and FITC labeledanti-mouse H-2Dd (110606, BioLegend), and expression levels of MHC classI on cancer cells were determined with flow cytometry. Dead cells hadbeen stained with a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kitpurchased from Thermo Fisher Scientific, and excluded from the analysis.The mean fluorescence intensity (MFI) of MHC class I was calculated, andMFI for cells treated with an Isotype control was subtracted from theMFI for stained cells, and the resulting value was used as adjusted MFI.Comparison between the control group and the compound (A) group wasperformed by using Dunnett's test, P values were expressed as numericalvalues to the fourth decimal place, and values of P<0.05 (two-tailedtest) were regarded as significant differences.

The results are shown in FIG. 17.

It was found that the expression level of MHC class I on CT26.WT-hHER2cells was significantly elevated by the compound (A) (FIG. 17). Hence,it was suggested that the compound (A) activates antitumor immunitythrough promoting elevation of the expression level of MHC class I oncancer cells.

Evaluation Example 9: Antitumor Test Using Nude Mice

Mouse: 6-week-old female BALB/c-nu mice (CAnN.Cg-Foxn1[nu]/CrlCrlj[Foxn1nu/Foxn1nu]) (Charles River Laboratories Japan, Inc.) weresubjected to experiment.

Assay and calculation expression: The major axis and minor axis of atumor were measured twice a week by using an electronic digital caliper(CD15-CX, Mitutoyo Corp.), and the tumor volume (mm³) was calculated.The calculation expression is as shown below.Tumor volume (mm³)=0.5×Major axis (mm)×[Minor axis (mm)]²

From the viewpoint of animal testing ethics, individuals whose tumorvolume exceeded 3000 mm³ were euthanized.

The antibody-drug conjugate (1) was intravenously administered to thetail vein of each mouse at a dose of 10 mg/kg. CT26.WT-hHER2 cells weresuspended in physiological saline, and 5.0×10⁶ cells were subcutaneouslytransplanted to the right axilla of each BALB/c-nu mouse, and the micewere randomly grouped 3 days thereafter (Day 0). The antibody-drugconjugate (1) was intravenously administered to the tail vein of eachmouse at a dose of 10 mg/kg on Days 0 and 7, twice in total. A groupwith administration of the solvent for the antibody-drug conjugate (1)was established as a control group. The number of mice in each group was12, and tumor volumes were measured until Day 13.

The results are shown in FIG. 18. The ordinate depicts tumor volumes(mm³) and the abscissa depicts days from the day of initialadministration. The antitumor effect by administration of theantibody-drug conjugate (1), which had been found for BALB/c mice, wasnot found for the BALB/c-nu mice. From the finding that the number of Tcells and that of B cells were reduced and the functions were impairedin the BALB/c-nu mice, it was inferred that these cells play animportant role for the antitumor effect of the antibody-drug conjugate(1).

Evaluation Example 10: Antitumor Test

In the same manner as in Evaluation Example 1, transition of tumorvolume in mice with subcutaneously transplanted CT26.WT-hHER2 cells wasdetermined for an antibody-drug conjugate (1)-administered group, acontrol antibody-drug conjugate-administered group, and a control group.

The control antibody-drug conjugate (Drug-to-Antibody Ratio: 7.8), usinga human IgG1 antibody which binds to molecules other than those derivedfrom mice and humans, was diluted with special solvent for use. Groupingwas performed 5 days after the transplantation (Day 0). The controlantibody-drug conjugate or antibody-drug conjugate (1) was intravenouslyadministered to the tail vein of each mouse at a dose of 10 mg/kg onDays 0 and 7, twice in total. The number of mice in each group was 10,and tumor volumes were measured until Day 10. Comparison onpharmaceutical effect between the control antibody-drug conjugate groupand the antibody-drug conjugate (1) group was performed by using theWilcoxon rank sum test, P values were expressed as numerical values tothe fourth decimal place, and values of P<0.05 (two-tailed test) wereregarded as significant differences.

The results are shown in FIG. 19. The ordinate depicts tumor volumes(mm³) and the abscissa depicts days from the day of initialadministration. On Day 10, the antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the controlantibody-drug conjugate group (P=0.0003). From the result, the antitumoreffect of the antibody-drug conjugate (1) was found to betarget-dependent.

Evaluation Example 11: Antitumor Test

In the same manner as in Evaluation Example 1, transition of tumorvolume in mice with subcutaneously transplanted EMT6-hHER2 cells wasdetermined for single administration groups with each of theantibody-drug conjugate (1) and an anti-PD-1 antibody (clone RMP1-14),and a combined administration group. The EMT6-hHER2 cells were preparedthrough transfection of a human HER2 gene into the mouse breast cancercell line EMT6 (CRL-2755) purchased from American Type CultureCollection by using a lentivirus vector. These cells were expressinghuman HER2 protein on their cell membranes. The EMT6-hHER2 cells weresuspended in physiological saline, and 1.0×10⁶ cells were subcutaneouslytransplanted to the right axilla of each 5-week-old BALB/c mouse, andthe mice were randomly grouped 4 days after the transplantation (Day 0).The antibody-drug conjugate (1) was intravenously administered to thetail vein of each mouse at a dose of 10 mg/kg once on Day 0. Ananti-PD-1 antibody (clone RMP1-14) was prepared with D-PBS(−) (WAKO),and intravenously administered to the tail vein of each mouse at a doseof 5.0 mg/kg on Days 0, 3, 7, and 10, four times in total. A combinedadministration group with the antibody-drug conjugate (1) and theanti-PD-1 antibody was established, and a group with administration ofthe special solvent for the antibody-drug conjugate (1) was establishedas a control group. The number of mice in each group was 11, and tumorvolumes were measured until Day 17. Comparison on pharmaceutical effectbetween the control group and each of the antibody-drug conjugate (1)group and the anti-PD-1 antibody group, and comparison on pharmaceuticaleffect between each of the antibody-drug conjugate (1) group and theanti-PD-1 antibody group and the combined administration group with bothagents were performed by using Dunnett's test (comparison among multiplegroups). P values adjusted for multiplicity were expressed as numericalvalues to the fourth decimal place, and values of P<0.05 (two-tailedtest) were regarded as significant differences.

The results are shown in FIG. 20. The ordinate depicts tumor volumes(mm³) and the abscissa depicts days from the day of initialadministration. On Day 17, the antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the control group(P<0.0001). The anti-PD-1 antibody group exhibited a significantlysuperior antitumor effect to the control group (P<0.0001). Further, thecombined administration group exhibited a significantly superiorantitumor effect to the antibody-drug conjugate (1) group (P=0.0136).The combined administration group exhibited a significantly superiorantitumor effect to the anti-PD-1 antibody group (P=0.0372). Weight losswas observed for none of the mice in all of the groups in this test.From the results, the antitumor effect of single administration of eachagent was confirmed, and it was further confirmed that such effect isdramatically enhanced through use of the two agents in combination.

Evaluation Example 12: Life Prolongation Test

In the same manner as in Evaluation Example 1, life-prolonging effectson mice with subcutaneously transplanted CT26.WT-hHER2 cells weredetermined for single administration groups with each of theantibody-drug conjugate (1) and an anti-PD-L1 antibody, and a combinedadministration group. An anti-PD-L1 antibody (clone 10F.9G2) waspurchased from Bio X Cell, and diluted with InVivoPure pH 6.5 DilutionBuffer (Bio X Cell) for use. The mice were randomly grouped 6 days afterthe transplantation (Day 0), and the antibody-drug conjugate (1) wasintravenously administered to the tail vein of each mouse at a dose of10 mg/kg on Days 0 and 7, twice in total. The anti-PD-L1 antibody wasintravenously administered to the tail vein of each mouse at a dose of 5mg/kg on Days 0 and 3, twice in total. A combined administration groupwith the antibody-drug conjugate (1) and the anti-PD-L1 antibody wasestablished, and a group with administration of the special solvent forthe antibody-drug conjugate (1) was established as a control group. Thenumber of mice in each group was 15, and tumor volumes were measureduntil Day 38. The day when estimated tumor volume exceeded 3000 mm³ (dayof euthanasia) was defined as the day of event occurrence (day ofdeath), and comparison on survival time between the control group andeach of the antibody-drug conjugate (1) group and the anti-PD-L1antibody group, and comparison on survival time between each of theantibody-drug conjugate (1) group and the anti-PD-L1 antibody group andthe combined administration group with both agents were performed byusing the Kaplan-Meier method/logrank test (comparison among multiplegroups). P values adjusted for multiplicity were expressed as numericalvalues to the fourth decimal place, and values of P<0.05 (two-tailedtest) were regarded as significant differences.

The results are shown in FIG. 21. The antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the control group(P=0.0069). The anti-PD-L1 antibody group exhibited a significantlysuperior antitumor effect to the control group (P=0.0037). Further, thecombined administration group exhibited a significantly superiorantitumor effect to the antibody-drug conjugate (1) group (P=0.0059).The combined administration group exhibited a significantly superiorantitumor effect to the anti-PD-L1 antibody group (P=0.0091). Weightloss was observed for none of the mice in all of the groups in thistest. From the results, the antitumor effect of single administration ofeach agent was confirmed, and it was further confirmed that such effectis dramatically enhanced through use of the two agents in combination.

Evaluation Example 13: Life Prolongation Test

EMT6-hHER2 cells were subcutaneously transplanted to mice in the samemanner as in Evaluation Example 11, and life-prolonging effects weredetermined for single administration groups with each of theantibody-drug conjugate (1) and an anti-PD-L1 antibody, and a combinedadministration group in the same manner as in Evaluation Example 12. Themice were randomly grouped 5 days after the transplantation (Day 0), andthe antibody-drug conjugate (1) was intravenously administered to thetail vein of each mouse at a dose of 10 mg/kg once on Day 0. Theanti-PD-L1 antibody was intravenously administered to the tail vein ofeach mouse at a dose of 5 mg/kg on Days 0 and 3, twice in total. Acombined administration group with the antibody-drug conjugate (1) andthe anti-PD-L1 antibody was established, and a group with administrationof the special solvent for the antibody-drug conjugate (1) wasestablished as a control group. The number of mice in each group wassix, and tumor volumes were measured until Day 60. Comparison onsurvival time between the control group and each of the antibody-drugconjugate (1) group and the anti-PD-L1 antibody group, and comparison onsurvival time between each of the antibody-drug conjugate (1) group andthe anti-PD-L1 antibody group and the combined administration group withboth agents were performed by using the Kaplan-Meier method/logrank test(comparison among multiple groups). P values adjusted for multiplicitywere expressed as numerical values to the fourth decimal place, andvalues of P<0.05 (two-tailed test) were regarded as significantdifferences.

The results are shown in FIG. 22. The antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the control group(P=0.0006). The anti-PD-L1 antibody group exhibited a significantlysuperior antitumor effect to the control group (P=0.0227). Further, thecombined administration group exhibited a significantly superiorantitumor effect to the antibody-drug conjugate (1) group (P=0.0039).Weight loss was observed for none of the mice in all of the groups inthis test. From the results, the antitumor effect of singleadministration of each agent was confirmed, and it was further confirmedthat such effect is dramatically enhanced through use of the two agentsin combination.

Evaluation Example 14: In Vivo CD4/8 Depletion Test

In the same manner as in Evaluation Example 1, transition of tumorvolume in mice with subcutaneously transplanted CT26.WT-hHER2 cells wasdetermined for single administration groups with each of theantibody-drug conjugate (1) and an anti-CD4 antibody, and a combinedadministration group, and for single administration groups with each ofthe antibody-drug conjugate (1) and an anti-CD8 antibody, and a combinedadministration group. The antibody-drug conjugate (1) was prepared toreach 10 mg/kg, and the anti-CD4 antibody (Bio X Cell, clone GK1.5) andanti-CD8 antibody (Bio X Cell, clone 53.6.7), each of which is adepletion antibody, were each prepared to reach 1 mg/mL with D-PBS(−)immediately before administration, and each of them was intravenouslyadministered to the tail vein of each mouse at a dose of 200 μg/head onDays 0 and 7. A group with administration of the special solvent for theantibody-drug conjugate (1) was established as a control group. Groupingwas performed 5 days after the transplantation (Day 0), tumor volumeswere measured until Day 11.

The results are shown in FIGS. 23 and 24. The tumor volume on Day 11 was651 mm³ for the antibody-drug conjugate (1) group, and 561 mm³ for thecombined administration group with the antibody-drug conjugate (1) andthe anti-CD4 antibody, suggesting that CD4-positive cells do notcontribute to the antitumor effect of the antibody-drug conjugate (1)(FIG. 23). The tumor volume on Day 11 was 651 mm³ for the antibody-drugconjugate (1) group, and 2247 mm³ for the combined administration groupwith the antibody-drug conjugate (1) and the anti-CD8 antibody,suggesting that CD8-positive cells contribute to the antitumor effect ofthe antibody-drug conjugate (1) (FIG. 24). Tumor volume had exceeded3000 mm³ in some individuals in the anti-CD4 antibody group and theanti-CD8 antibody group, and hence the individuals were euthanizedduring the test. Thus, each of the tumor growth curves ends at thetiming of euthanasia. In combination with the finding from the tumormodel using immunodeficient nude mice in Evaluation Example 9 that Tcells or B cells are partly involved in the pharmaceutical effect of theantibody-drug conjugate (1), the present results suggested thatCD8-positive T cells contribute to the antitumor effect of theantibody-drug conjugate (1).

Evaluation Example 15: Analysis of Intratumor T Cells

The fraction of CD8-positive T cells among intratumor living cells, thefraction of Granzyme B-positive cells among intratumor CD8-positive Tcells, the fraction of CD8-positive T cells being Granzyme B-positiveamong intratumor living cells, and the fraction of CD4-positive T cellsamong intratumor living cells were determined with flow cytometry forthe case that the antibody-drug conjugate (1) was administered to micewith subcutaneously transplanted CT26.WT-hHER2 cells. Cell suspensionswere prepared in the same manner as in Evaluation Example 6, and thenstaining was performed with a Pacific Blue labeled anti-mouse CD45antibody (103126, BioLegend), PE labeled anti-mouse CD3e antibody(553064, Becton Dickinson), PerCP/Cy5.5 labeled anti-mouse CD4 antibody(100434, BioLegend), PE-Cy 7 labeled anti-mouse CD8a antibody (552877,Becton Dickinson), and Alexa FluorR 647 labeled anti-human/mouseGranzyme B antibody (515405, BioLegend), and assayed with flowcytometry. Dead cells had been stained with a LIVE/DEAD Fixable Near-IRDead Cell Stain Kit purchased from Thermo Fisher Scientific, andexcluded from the analysis. Comparison between the control group and theantibody-drug conjugate (1) group was performed by using Student'st-test, P values were expressed as numerical values to the fourthdecimal place, and values of P<0.05 (two-tailed test) were regarded assignificant differences.

The results are shown in FIGS. 25 to 28. It was found that the fractionof CD45-, CD3-, CD8-positive cells (CD8-positive T cells) among livingcells significantly increased by administration of the antibody-drugconjugate (1) (FIG. 25). It was found that the fraction of GranzymeB-positive cells among CD8-positive T cells significantly increased byadministration of the antibody-drug conjugate (1) (FIG. 26). It wasfound that the fraction of CD8-positive T cells being GranzymeB-positive among living cells significantly increased by administrationof the antibody-drug conjugate (1) (FIG. 27). Although the fraction ofCD45-, CD3-, CD4-positive cells (CD4-positive T cells) among livingcells had an increasing tendency by administration of the antibody-drugconjugate (1), the difference was not significant (FIG. 28).

Thus, these results suggested that the antibody-drug conjugate (1)activates antitumor immunity by increasing the number of intratumorCD8-positive T cells and promoting activation thereof.

Evaluation Example 16: CD8 IHC Analysis

The number of CD8-positive cells per unit area in tumors was counted byusing IHC for the case that the antibody-drug conjugate (1) wasadministered to mice with subcutaneously transplanted CT26.WT-hHER2cells. In the same manner as in Evaluation Example 6, the control andthe antibody-drug conjugate (1) were administered, and the mice wereeuthanized 8 days after the administration. The number of mice in eachgroup was five, and tumors were excised from the intermediate number ofmice, namely, three mice, and each soaked in paraformaldehyde/phosphatebuffer to produce a paraffin block. Each paraffin block was stained withan anti-CD8 antibody (clone: 4SM16), and the sample image was taken byusing a NanoZoomer 2.0-HT (Hamamatsu Photonics K.K.), and the wholeregion of tissue was analyzed by using the image analysis softwareTissue Studio3.0 (Definiens).

The results are shown in FIGS. 29 and 30. It was found that the numberof CD8-positive cells per unit area in tumors tended to increase by theaction of the antibody-drug conjugate (1).

Evaluation Example 17: Analysis of In Vitro Cancer Cells

Expression levels of MHC class I were determined for cancer cellstreated with different compounds. In the same manner as in EvaluationExample 8, each of the compound (A), DM1-SMe, DM4-SMe (J. Med. Chem.(2014), 57, 16, 6949-6964), and MMAE (Molecular Cancer Therapeutics(2011), 10, 9, 1728-1739) was added to a concentration of 20 nM, 100 nM,or 500 nM, and the expression level of MHC class I on cancer cells wasdetermined with flow cytometry. The experiment was performed intriplicate. Comparison between the control group and each of the agentgroups at each concentration was performed by using Dunnett's test, Pvalues were expressed as numerical values to the fourth decimal place,and values of P<0.05 (two-tailed test) were regarded as significantdifferences (***: P<0.001, **: P<0.01).

The results are shown in FIG. 31. At the concentrations examined (20 nM,100 nM, 500 nM), all of the agents evaluated were found to significantlyelevate the expression level of MHC class I on CT26.WT-hHER2 cells ascompared with the control group. Among them, the compound (A) was foundto exhibit the maximum elevation effect on expression of MHC class I onCT26.WT-hHER2 cells.

Evaluation Example 18: Antitumor Test

In the same manner as in Evaluation Example 11, transition of tumorvolume in mice with subcutaneously transplanted EMT6-hHER2 cells wasdetermined for single administration groups with each of theantibody-drug conjugate (1) and an anti-CTLA-4 antibody, and a combinedadministration group. An anti-CTLA-4 antibody (clone 9H10) was purchasedfrom Bio X Cell, and diluted with D-PBS(−) for use. Grouping wasperformed 5 days after the transplantation (Day 0). The antibody-drugconjugate (1) was intravenously administered to the tail vein of eachmouse at a dose of 10 mg/kg once on Day 0. The anti-CTLA-4 antibody wasintravenously administered to the tail vein of each mouse at a dose of5.0 mg/kg on Days 0, 3, and 7, three times in total. A combinedadministration group with the antibody-drug conjugate (1) and theanti-CTLA-4 antibody was established, and a group with administration ofthe special solvent for the antibody-drug conjugate (1) was establishedas a control group. The number of mice in each group was 10, and tumorvolumes were measured until Day 14. Comparison on pharmaceutical effectbetween the control group and each of the antibody-drug conjugate (1)group and the anti-CTLA-4 antibody group, and comparison onpharmaceutical effect between each of the antibody-drug conjugate (1)and the anti-CTLA-4 antibody group and the combined administration groupwith both agents were performed by using Dunnett's test (comparisonamong multiple groups). P values adjusted for multiplicity wereexpressed as numerical values to the fourth decimal place, and values ofP<0.05 (two-tailed test) were regarded as significant differences.

The results are shown in FIG. 32. The ordinate depicts tumor volumes(mm³) and the abscissa depicts days from the day of initialadministration. On Day 14, the antibody-drug conjugate (1) groupexhibited a significantly superior antitumor effect to the control group(P=0.0011). The anti-CTLA-4 antibody group exhibited a significantlysuperior antitumor effect to the control group (P=0.0006). Further, thecombined administration group exhibited a significantly superiorantitumor effect to the antibody-drug conjugate (1) group (P=0.0115).The combined administration group exhibited a significantly superiorantitumor effect to the anti-CTLA-4 antibody group (P=0.0309). From theresults, the antitumor effect of single administration of each agent wasconfirmed, and it was further confirmed that such effect is dramaticallyenhanced through use of the two agents in combination.

From the above experimental results, the antibody-drug conjugateaccording to the present invention was revealed to exhibit adramatically excellent antitumor effect through being administered incombination with an immune checkpoint inhibitor. In addition, theantibody-drug conjugate according to the present invention wasdemonstrated to have an antitumor immunity-activating effect.Accordingly, the antibody-drug conjugate can provide a pharmaceuticalcomposition and therapeutic method superior in antitumor effect andsafety.

Free Text of Sequence Listing

SEQ ID NO: 1—Amino acid sequence of a heavy chain of the humanizedanti-HER2 antibody

SEQ ID NO: 2—Amino acid sequence of a light chain of the humanizedanti-HER2 antibody

The invention claimed is:
 1. A therapeutic method wherein anantibody-drug conjugate and an immune checkpoint inhibitor areadministered in combination to a subject in need thereof, and theantibody-drug conjugate is an antibody-drug conjugate in which adrug-linker represented by the following formula:

wherein A represents the connecting position to an antibody, isconjugated to the antibody via a thioether bond.
 2. The therapeuticmethod according to claim 1, wherein the antibody in the antibody-drugconjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2antibody, or an anti-B7-H3 antibody.
 3. The therapeutic method accordingto claim 2, wherein the antibody in the antibody-drug conjugate is ananti-HER2 antibody.
 4. The therapeutic method according to claim 2,wherein the anti-HER2 antibody is an antibody comprising a heavy chainconsisting of an amino acid sequence consisting of amino acid residues 1to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acidsequence consisting of amino acid residues 1 to 214 of SEQ ID NO:
 2. 5.The therapeutic method according to claim 2, wherein the anti-HER2antibody is an antibody comprising a heavy chain consisting of the aminoacid sequence represented by SEQ ID NO: 1 and a light chain consistingof the amino acid sequence represented by SEQ ID NO:
 2. 6. Thetherapeutic method according to claim 1, wherein the average number ofunits of the drug-linker conjugated per antibody molecule in theantibody-drug conjugate is in the range of from 2 to
 8. 7. Thetherapeutic method according to claim 1, wherein the average number ofunits of the drug-linker conjugated per antibody molecule in theantibody-drug conjugate is in the range of from 7 to
 8. 8. Thetherapeutic method according to claim 1, wherein the average number ofunits of the drug-linker conjugated per antibody molecule in theantibody-drug conjugate is in the range of from 7.5 to
 8. 9. Thetherapeutic method according to claim 1, wherein the immune checkpointinhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or ananti-CTLA-4 antibody.
 10. The therapeutic method according to claim 9,wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. 11.The therapeutic method according to claim 9, wherein the immunecheckpoint inhibitor is an anti-PD-L1 antibody.
 12. The therapeuticmethod according to claim 9, wherein the immune checkpoint inhibitor isan anti-CTLA-4 antibody.
 13. The therapeutic method according to claim1, wherein the antibody-drug conjugate and the immune checkpointinhibitor are separately contained as active components in differentformulations, and are administered simultaneously or at different times.14. The therapeutic method according to claim 1, wherein theantibody-drug conjugate and the immune checkpoint inhibitor arecontained as active components in a single formulation and administered.15. The therapeutic method according to claim 1, wherein the method isfor treating cancer.
 16. The therapeutic method according to claim 15,wherein the cancer is at least one selected from the group consisting oflung cancer, urothelial cancer, colorectal cancer, prostate cancer,ovarian cancer, pancreatic cancer, breast cancer, bladder cancer,gastric cancer, esophagogastric junction adenocarcinoma,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma,neuroepithelial tissue tumor, nerve sheath tumor, head-and-neck cancer,skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer,mesothelioma, Paget's disease, and sarcoma.
 17. The therapeutic methodaccording to claim 16, wherein the cancer is colorectal cancer.
 18. Thetherapeutic method according to claim 16, wherein the cancer is breastcancer.
 19. The therapeutic method according to claim 1, wherein theantibody-drug conjugate has an antitumor immunity-activating effect. 20.The therapeutic method according to claim 1, wherein the antibody-drugconjugate has at least one effect selected from the group consisting of:(1) a promoting effect on growth of intratumor CD8-positive T cells; and(2) an activating effect on intratumor CD8-positive T cells.
 21. Thetherapeutic method according to claim 1, wherein the antibody-drugconjugate has a promoting effect on the formation of immune memoryagainst tumor.
 22. The therapeutic method according to claim 21, whereinthe tumor is expressing an antigen for the antibody in the antibody-drugconjugate.
 23. The therapeutic method according to claim 21, wherein apart of the cells of the tumor are not expressing an antigen for theantibody in the antibody-drug conjugate.
 24. The therapeutic methodaccording to claim 1, wherein the antibody-drug conjugate has at leastone effect selected from the group consisting of: (1) a promoting effecton increase of the number of dendritic cells in a tumor; (2) anactivating effect on dendritic cells; and (3) a promoting effect onelevation of the expression level of WIC class I on cancer cells. 25.The therapeutic method according to claim 1, wherein the immunecheckpoint inhibitor deactivates an immunosuppression signal generatedthrough elevation of the expression level of PD-L1 on cancer cellspromoted by the antibody-drug conjugate, and thereby the antibody-drugconjugate exhibits a higher antitumor effect.